Recombinant infectious bovine rhinotrocheitis virus

ABSTRACT

The present invention provides a hybrid, nonprimate herpesvirus comprising DNA which includes a sequence essential for viral replication of the hybrid, nonprimate herpesvirus, at least a portion of which is present in a sequence essential for replication of a naturally-occurring nonprimate herpesvirus and at least one foreign DNA sequence. 
     Also provided is an attenuated, nonprimate herpesvirus comprising DNA which includes a sequence essential for viral replication of the attenuated, nonprimate herpesvirus, at least a portion of which is present in a sequence essential for replication of a naturally-occurring nonprimate herpesvirus, from which at least a portion of a repeat sequence has been deleted. 
     Also provided are vaccines comprising the viruses of the invention and methods of immunizing animals against various disease.

The subject application is a continuation of U.S. Ser. No. 08/117,633,filed Sep. 7, 1993; now abandoned which is a continuation of U.S. Ser.No. 07/914,057, filed Jul. 13, 1992, now abandoned; which is acontinuation of U.S. Ser. No. 07/696,262, filed April 30, 1991, nowabandoned; which is a continuation of U.S. Ser. No. 06/933,107, filedNov. 20, 1986, now abandoned; which is a continuation-in-part of U.S.Ser. No. 06/902,877, filed Sep. 2, 1986, now abandoned which is acontinuation-in-part of U.S. Ser. No. 06/887,140, filed Jul. 17, 1986,now abandoned; which is a continuation-in-part of U.S. Ser. No.06/823,102, filed Jan. 27, 1986, now U.S. Pat. No. 5,068,192, issuedNov. 26, 1991, which is a continuation-in-part of U.S. Ser. No.06/773,430, filed Sep. 6, 1988, now U.S. Pat. No. 4,879,737, issued Oct.31, 1989, which are hereby incorporated by reference into the presentapplication.

BACKGROUND OF THE INVENTION

Within this application several publications are referenced by Arabicnumerals within parentheses. Full citations for these references may befound at the end of the specification immediately preceding the claims.The disclosures of these publications in their entirety are herebyincorporated by reference into this application in order to more fullydescribe the state of the art to which this invention pertains.

The advent of recombinant DNA techniques has made it possible tomanipulate the naturally occurring DNA sequences within an organism (thegenome) in order to change in some manner the functions of the organismthrough genetic engineering. The present invention concerns organismsdefined as viruses that infect animals and contain DNA as their geneticmaterial; specifically viruses belonging to the herpesvirus group(herpesviruses) (28). This group of viruses comprise a number ofpathogenic agents that infect and cause disease in a number of targetspecies: swine, cattle, chickens, horses, dogs, cats, etc. Eachherpesvirus is specific for its host species, but they are all relatedin the structure of their genomes, their mode of replication, and tosome extent in the pathology they cause in the host animal and in themechanism of the host immune response to the virus infection.

The types of genetic engineering that have been performed on theseherpesviruses consist of cloning parts of the virus DNA into plasmids inbacteria, reconstructing the virus DNA while in the cloned state so thatthe DNA contains deletions of certain sequences, and furthermore addingforeign DNA sequences either in place of the deletions or at sitesremoved from the deletions. The usual method is to make insertions ofthe foreign DNA into the viral sequences, although the foreign DNA couldbe attached to the end of the viral DNA as well. One utility of theaddition of foreign sequences is achieved when the foreign sequenceencodes a foreign protein that is expressed during viral infection ofthe animal. A virus with these characteristics is referred to as avector, because it becomes a living vector which will carry and expressthe foreign protein in the animal. In effect it becomes an elaboratedelivery system for the foreign protein.

The prior art for this invention stems first from the ability to cloneand analyze DNA while in the bacterial plasmids. The techniques that areavailable for the most part are detailed in Maniatis et al. (1). Thispublication gives state-of-the-art general recombinant DNA techniques.

The application of recombinant DNA techniques to animal viruses has arelatively recent history from about 1980. The first viruses to beengineered have been the smallest ones--the papovaviruses. The virusescontain 3000-4000 base pairs (bp) of DNA in their genome. Their smallsize makes analysis of their genomes relatively easy and in fact most ofthe ones studied (SV40, polyoma, bovine papilloma) have been entirelysequenced. Because these virus particles are small and cannotaccommodate much extra DNA, and because their DNA is tightly packed withessential sequences (that is, sequences required for replication), ithas not been possible to engineer these viruses as live vectors forforeign gene expression. Their entire use in genetic engineering hasbeen as defective replicons for the expression of foreign genes inanimal cells in culture (roughly analogous to plasmids in bacterialsystems) or to their use in mixed populations of virions in which wildtype virus acts as a helper for the virus that has replaced an essentialpiece of DNA with a foreign gene. The studies on papovaviruses do notsuggest or teach the concept of living virus vectors as delivery systemsfor host animals.

The next largest DNA animal viruses are the adenoviruses. In theseviruses there is a small amount of nonessential DNA that can be replacedby foreign sequences. The only foreign genes that seem to have beenexpressed in adenoviruses are the T-antigen genes from papovaviruses(2,3,4,5), and the herpes simplex virus thymidine kinase gene (29). Itis possible, given this initial success, to envision the insertion ofother small foreign genes into adenoviruses. However the techniques usedin adenoviruses do not teach how to obtain the same result withherpesviruses. In particular, these results do not identify thenonessential regions in herpesviruses wherein foreign DNA can beinserted, nor do they teach how to achieve the expression of the foreigngenes in herpesviruses, e.g. which promoter signals and terminationsignals to use. Another group of animal viruses that have beenengineered are the poxviruses. One member of this group, vaccinia, hasbeen the subject of much research on foreign gene expression. Poxvirusesare large DNA-containing viruses that replicate in the cytoplasm ofinfected cells. They have a structure that is very unique amongviruses--they do not contain any capsid that is based upon icosahedralsymmetry or helical symmetry. In theorizing on the origin of viruses,the poxviruses are the most likely ones to have originated frombacterial-like microorganisms through the loss of function anddegeneration. In part due to this uniqueness, the advances made in thegenetic engineering of poxviruses cannot be directly extrapolated toother viral systems, including herpesviruses. Vaccinia recombinant virusconstructs have been made in a number of laboratories that express thefollowing inserted foreign genes: herpes simplex virus thymidine kinasegene (6,7), hepatitis B surface antigen (8,9,30), herpes simplex virusglycoprotein D gene (8,30), influenza hemagglutinin gene (10, 11),malaria antigen gene (12), and vesicular stomatitis glycoprotein G gene(13). The general overall features of the vaccinia recombinant DNA workare similar to the techniques used for all the viruses, especially asthey relate to the techniques in reference (1). However in detail, thevaccinia techniques do not teach how to engineer herpesviruses. VacciniaDNA is not infectious, so the incorporation of foreign DNA must involvean infection/transfection step that is not appropriate to other viruses,and vaccinia has unique stability characteristics that make screeningeasier. In addition, the signal sequence used by promoters in vacciniaare unique and will not work in other viruses. The utility of vacciniaas a vaccine vector is in question because of its close relationship tohuman smallpox and its known pathogenicity to humans. The use ofhost-specific herpesviruses promises to be a better solution to animalvaccination.

Among the herpesviruses, only herpes simplex of humans and, to a limitedextent, herpes saimiri of monkeys have been engineered to containforeign DNA sequences previous to this disclosure. The earliest work onthe genetic manipulation of herpes simplex virus involved the rescue oftemperature sensitive mutants of the virus using purified restrictionfragments of DNA (14). This work did not involve cloning of the DNAfragments into the viral genome. The first use of recombinant DNA tomanipulate herpes simplex virus involved cloning a piece of DNA from theL-S junction region into the unique long region of the DNA, specificallyinto the thymidine kinase gene (15). This insert was not a foreign pieceof DNA, rather it was a naturally occurring piece of herpesvirus DNAthat was duplicated at another place in the genome. This piece of DNAwas not engineered to specifically express any protein, and thus it didnot teach how to express protein in herpesviruses. The manipulation ofherpes simplex next involved the creation of deletions in the virusgenome by a combination of recombinant DNA and thymidine kinaseselection. The first step was to make a specific deletion of thethymidine kinase gene (16). The next step involved the insertion of thethymidine kinase gene into the genome at a specific site, and then thethymidine kinase gene and the flanking DNA at the new site were deletedby a selection against thymidine kinase (17). In this manner herpessimplex alpha-22 gene has been deleted (17). In the most recentrefinement of this technique, a 15,000 bp sequence of DNA has beendeleted from the internal repeat of herpes simplex virus (18).

The insertion of genes that encode protein into primate herpesviruseshave involved seven cases: the insertion of herpes simplex glycoproteinC back into a naturally occurring deletion mutant of this gene in herpessimplex virus (19); the insertion of glycoprotein D of herpes simplextype 2 into herpes simplex type 1 (20), again with no manipulation ofpromoters since the gene is not really `foreign`; the insertion ofhepatitis B surface antigen into herpes simplex virus under the controlof the herpes simplex ICP4 promoter (21); and the insertion of bovinegrowth hormone into herpes saimiri virus with an SV40 promoter that infact didn't work in that system (an endogenous upstream promoter servedto transcribe the gene) (22). Two additional cases of foreign genes(chicken ovalbumin gene and Epstein-Barr virus nuclear antigen) havebeen inserted into herpes simplex virus (31), and glycoprotein X ofpseudorabies virus has been inserted into herpes simplex virus (33).

These limited cases of deletion and insertion of genes intoherpesviruses demonstrate that it is possible to genetically engineerherpesvirus genomes by recombinant DNA techniques. The methods that havebeen used to insert genes involve homologous recombination between theviral DNA cloned on plasmids and purified viral DNA transfected into thesame animal cell. In aggregate this is referred to as the homologousrecombination technique. This technique with minor modifications hasbeen adaptable to other herpesviruses that we have engineered. However,the extent to which one can generalize the location of the deletion andthe sites for insertion of foreign genes is not obvious from theseprevious studies. Furthermore, it is also not obvious that non-primateherpesviruses are amenable to the same techniques as the primateherpesviruses, and that one could establish a targeted approach to thedeletion, insertion, and expression of foreign genes.

One subject of this invention is a vaccine for pseudorabies virus(herpesvirus suis, suid herpesvirus 1, or Aujesky's disease virus)disease of swine. Swine are the natural host of pseudorabies virus inwhich infection in older animals is commonly inapparent but may becharacterized by fever, convulsions, and death particularly in youngeranimals. Pseudorabies also infects cattle, sheep, dogs, cats, ferrets,foxes, and rats (39) where the infection usually results in death. Deathis usually preceded by intense pruritus, mania, encephalitis, paralysis,and coma. Traditional live vaccines are available for use in swine, butthey are lethal for the other animals. An improved vaccine forpseudorabies would induce a more reliable immune response in swine,would be specifically attenuated to be incapable of reversion tovirulence, and would not cause disease in other hosts.

Pseudorabies virus, an alpha-herpesvirus of swine, has a genome of classD (23); that is it contains two copies of a single repeat region, onelocated between the unique long and unique short DNA region and one atthe terminus of the unique short region (see FIG. 1). Herpes simplexvirus is an alpha-herpesvirus with a class E genome (28); that is itcontains two copies of each of two repeats. Herpes saimiri is agamma-herpesvirus with a class B genome (28) and thus is less relatedstructurally to pseudorabies than is herpes simplex virus.

Pseudorabies virus has been studied using the tools of molecular biologyincluding the use of recombinant DNA techniques. BamHI, KpnI, and BgIIIrestriction maps of the virus genome have been published (24, 27). DNAtransfection procedures have been utilized to rescue temperaturesensitive and deletion mutants of the virus by the homologousrecombination procedure (24). There are two examples of deletions thathave been made in the pseudorabies virus genome--one is a thymidinekinase gene deletion (25), also disclosed in Pat. No. 4,514,497 entitled"Modified Live Pseudorabies Viruses". This patent teaches thymidinekinase deletions only and does not suggest other attenuating deletions,nor does it suggest insertion of foreign DNA sequences. The otherreference involves the deletion of a small DNA sequence around a HindIIIrestriction site in the repeat region (26) upon which European PatentPublication No. 0141458, published on May 15, 1985, corresponding toEuropean Patent Application No. 84201474.8, filed on Oct. 12, 1984 isbased. This patent application does not teach or suggest attenuatingdeletions nor does it teach or suggest the insertion of DNA sequencesinto pseudorabies virus.

Other relevant pseudorabies literature disclosed herein, concerns thepresence of naturally-occurring deletions in the genome of two vaccinestrains of pseudorabies viruses (27). These deletions are responsible,at least in part, for the attenuated nature of these vaccines. Suchnaturally-occurring deletions do not teach methods for making thesedeletions starting with wild type pseudorabies virus DNA, nor do theysuggest other locations at which to make attenuating deletions. Thereare no examples of naturally-occurring insertions of foreign DNA inherpesviruses.

Infectious bovine rhinotracheitis (IBR) virus, an alpha-herpesvirus witha class D genome, is an important pathogen of cattle. It has beenassociated with respiratory, ocular, reproductive, central nervoussystem, enteric, neonatal and dermal diseases (39). Cattle are thenormal hosts of IBR virus, however it also infects goats, swine, waterbuffalo, wildebeest, mink and ferrets. Experimental infections have beenestablished in muledeer, goats, swine, ferrets and rabbits (40).

Conventional modified live virus vaccines have been widely used tocontrol diseases caused by IBR. These vaccine viruses may revert tovirulence, however. More recently, killed virus IBR vaccines have beenused, but their efficacy appears to be marginal.

IBR has been analyzed at the molecular level as reviewed in (41). Arestriction map of the genome is available in this reference, which willaid in the genetic engineering of IBR according to the methods providedby the present invention. No evidence has been presented that IBR hasbeen engineered to contain a deletion or an insertion of foreign DNA.

Marek's disease virus (MDV) causes fowl paralysis, a commonlymphoproliferative disease of chickens. The disease occurs mostcommonly in young chickens between 2 and 5 months of age. The prominantclinical signs are progressive paralysis of one or more of theextremeties, incoordination due to paralysis of legs, drooping of thelimb due to wing involvement, and a lowered head position due toinvolvement of the neck muscles. In acute cases, severe depression mayresult. In the case of highly oncogenic strains, there is characteristicbursal and thymic atrophy. In addition, there are lymphoid tumorsaffecting the gonads, lungs, liver, spleen, kidney and thymus (39).

All chicks are vaccinated against MDV at one day of age to protect thechick against MDV for its lifetime. One vaccine method for MDV involvesusing turkey herpesvirus (HVT). It would be advantageous to incorporateother antigens into this vaccination at one day of age, but efforts tocombine vaccines have not proven satisfactory to date due to competitionand immunosuppression between pathogens. The multivalent vaccinesengineered in this invention are a novel way to simultaneously vaccinateagainst a number of different pathogens.

A restriction map of both MDV (45) and HVT (36) are available in theliterature. There is no evidence to suggest that anyone has successfullycreated a deletion or insertion of foreign DNA into MDV or HVT prior tothis disclosure.

Other herpesviruses contemplated to be amenable to these procedures arefeline herpesvirus (FHV), equine herpesvirus (EHV), and canineherpesvirus (CHV). These pathogens cause disease in each of theirrespective hosts. Feline herpesvirus causes feline rhinotracheitis, anacute upper respiratory tract infection characterized by fever,pronounced sneezing, nasal and lacrimal secretions, and depression. Thevirus may cause corneal ulceration and abortion. The nasal passages andturbinates show focal necrosis, and the tonsils are enlarged andhemorrhagic. Equine herpesvirus causes rhinopneumonitis, abortion,exanthema of the genitals and occasionally neurologic disease. The acutedisease is characterized by fever, anorexia and a profuse, serous nasaldischarge. The neurologic symptoms, when present, consist of ataxia,weakness and paralysis. Canine herpesvirus causes severe illness inyoung puppies, where mortality may reach 80%. The disease ischaracterized by viremia, anorexia, respiratory illness, abdominal pain,vomiting and incessant crying. Generally, there is no fever. Theprincipal lesions are disseminated necrosis and hemorrhages in thekidneys, liver and lungs.

The molecular biology of the feline, equine and canine herpesviruses arein their initial phases. Partial restriction maps are available forequine herpesvirus, and in progress in at least one lab for the felineherpesvirus. Beyond this type of genome analysis, no evidence for thedeletion or insertion of foreign genes into these viruses is available.

The present invention involves the use of genetically engineeredherpesviruses to protect animals against disease. It is not obviouswhich deletions in herpesviruses would serve to attenuate the virus tothe proper degree. Even testing vaccine candidates in animal models,e.g. mice, does not serve as a valid predictor of the safety andefficacy of the vaccine in the target animal species, e.g. swine.

SUMMARY OF THE INVENTION

The present invention provides a hybrid, nonprimate herpesviruscomprising DNA which includes a sequence essential for viral replicationof the hybrid, non-primate herpesvirus, at least a portion of which ispresent in a sequence essential for replication of a naturally-occurringnonprimate herpesvirus and at least one foreign DNA sequence.

Also provided is an attenuated, nonprimate herpesvirus comprising DNAwhich includes a sequence essential for viral replication of theattenuated, nonprimate herpesvirus, at least a portion of which ispresent in a sequence essential for replication of a naturally-occurringnonprimate herpesvirus, from which at least a portion of a repeatsequence has been deleted.

The present invention further provides an attenuated, hybrid, nonprimateherpesvirus. This virus comprises DNA which includes a sequenceessential for viral replication of the attenuated, hybrid, nonprimateherpesvirus, at least a portion of which is present in a sequenceessential for replication of a naturally-occurring nonprimateherpesvirus and at least one foreign DNA sequence.

BRIEF DESCRIPTION OF THE FIGURES FIGS. 1A AND 1B DETAILS OF WILD TYPESHOPE STRAIN PRV

1A. Diagram of PRV genomic DNA showing the unique long region (UL), theunique short region (US), the internal repeat region (IR), and theterminal repeat region (TR).

1B. BamHI restriction enzyme map of PRV. Fragments are numbered in orderof decreasing size.

FIGS. 2A-2C Details of S-PRV-004 Construction and Map Data

2A. Detailed map of BamHI #8' and #8. The location of the internalrepeat (IR) region is shown.

2B. Detailed map of BamHI #8'-TK-8 fragment ultimately present in therecombinant virus.

2C. Diagram of the S-PRV-004 DNA genome showing the location of theHSV-1 TK gene inserted into the junction region between the UL and IRregions.

Restriction Enzyme Legend: B=BamHI; K=KpnI; N=NdeI; P=PvuII; S=StuI.

FIGS. 3A-3C Details of S-PRV-005 Construction and Map Data.

3A. Detailed map of BamHI #5. The HSV-1 TK gene fused to the HSV-1 ICP4promoter is shown on a PvuII fragment.

3B. Detailed map of BamHI #5 after the insertion of the TK geneconstruct.

3C. Diagram of the S-PRV-005 DNA genome showing the location of the TKgene inserted into both copies of BamHI #5 in the repeat region of thegenome and the creation of new deletions.

Restriction Enzyme Legend: B=BamHI; H=HindIII; Hp=HpaI; K=KpnI; P=PvuII;X=XbaI.

FIGS. 4A-4D Construction of the Foreign DNA Insert Used in S-PRV-010.

4A. Diagram of the relevant portion of pJF751 that contains the lac Z(beta-galactosidase) gene. The position of the TAA termination codon forthe polypeptide is indicated.

4B. Diagram of the promoter sequence from the HSV-1 TK gene.

4C. Diagram of the RsaI fragment of the TK gene now with BamHI modifiedends.

4D. Diagram of the final plasmid that contained the lac Z gene fused tothe HSV-1 TK promoter.

Restriction Enzyme legend: B=BamHI; Ba=BalI; Bc=BclI; Bg=BglII;H=HindIII; Ha=HaeIII; N=NdeI; R=RsaI; X=XbaI.

FIGS. 5A-5C Details of S-PRV-010 Construction and Map Data.

5A. Detailed map of BamHI #5. The lac Z gene (beta-galactosidase) fusedto the HSV-1 TK promoter is shown on an XbaI fragment (see FIGS. 4A-4D).The position of the deletion in S-PRV-002 is shown.

5B. Detailed map of BamHI #5 after the insertion of the lac Z geneconstruct.

5C. Diagram of the S-PRV-010 genome DNA showing the location of the lacZ gene into both copies of BamHI #5 in the repeat region of the genome.

Restriction Enzyme Legend: B=BamHI; Bc=BclI; H=HindIII; Hp=HpaI; K=KpnI;N=NdeI; X=XbaI.

FIGS. 6A-6C Details of S-PRV-007 Construction and Map Data.

6A. Detailed map of BamHI #5 from S-PRV-005.

6B. Detailed map of BamHI #5 after the substitution of the TK gene withthe swine rotavirus gp38 gene.

6C. Diagram of the S-PRV-007 DNA genome showing the location of the gp38gene inserted into both copies of BamHI #5 in the repeat regions of thegenome.

Restriction Enzyme Legend: B=BamHI; H=HindIII; K=KpnI. FIG. 7Construction of the Foreign DNA Insert Used in S-PRV-007.

FIGS. 8A-8C Details of S-PRV-012 Construction and Map Data.

8A. Detailed map of PRV extending from BamHI #10 through BamHI #7.

8B. Detailed map of PRV extending from BamHI #10 through BamHI #7 afterthe insertion of the TK gene into the recombinant virus.

8C. Diagram of the S-PRV-012 DNA genome showing the location of the TKgene inserted into the gpX region and the creation of a deletion thatremoves most of the coding region of the gpX gene and renders the virusunable to synthesize the gpX polypeptide.

Restriction Enzyme Legend: B=BamHI; K=KpnI; N=NdeI; P=PvuII; Ps=PstI;S=StuI.

FIGS. 9A-9E Details of S-PRV-013. S-PRV-014, and S-PRV-016 Constructionand Map Data.

9A. Detailed map of PRV extending from BamHI #10 through BamHI #7.

9B. Detailed map of PRV extending from BamHI #10 through BamHI #7 afterthe insertion of the lac Z gene into the recombinant virus.

9C. Diagram of the S-PRV-013 DNA genome showing the location of the lacZ gene inserted into the gpX region and the creation of a deletion thatremoved most of the coding region of the gpX gene and rendered the virusunable to synthesize the gpX polypeptide. Other deletions in the TKregion and repeat regions are shown by (▴).

9D. Diagram of the S-PRV-014 DNA genome showing the location of the lacZ gene inserted into the gpX region and the creation of a deletion thatremoved most of the coding region of the gpX gene and rendered the virusunable to synthesize the gpX polypeptide. There are no other deletionsin this virus.

9E. Diagram of the S-PRV-016 DNA genome showing the location of the lacZ gene inserted into the gpX region and the creation of a deletion thatremoved most of the coding region of the gpX gene and rendered the virusunable to synthesize the gpX polypeptide. Other deletions in the repeatregions are shown by (▴).

Restriction Enzyme Legend: B=BamHI; Ba=BalI; K=KpnI; N=NdeI; Ps=PstI;S=StuI.

FIG. 10A and 11B Swine rotavirus gp38 Gene Sequence in pSY565.

FIG. 11A and 11B Swing parvovirus B gene sequence in pSY875.

FIG. 12 Swine parvovirus B gene construction with signal sequence

A. pSY864 which contains the B gene from AccI at nucleotide #391 to RsaIsite at nucleotide #2051 cloned between the BamHI site in BamHI #10 andthe NdeI site in BamHI #7.

B. pSY957 which contains the SailI fragment from pSY864 cloned into apolylinker in pSP65 so that XbaI sites flank the insert.

Legend: pSP=E. coli plasmid; PRV=pseudorabies virus DNA; PPV=porcineparvovirus DNA; Pv=PvuII; RV=EcoRV; Ps=PstI; B=BamHI; A=AccI; R=RsaI;N=NdeI; Sa=SalI; Sm=SmaI; S=StuI; X=XbaI; gpX pro=glycoprotein Xpromoter; gpX pA=glycoprotein X polyadenylation signal sequences.

FIG. 13A-13 C Details of S-PRV-020 Construction and Map Data

13A. Detailed map of PRV extending from BamHI #10 through BamHI #7showing the parvovirus B gene that will replace the gpX gene.

13B. Detailed map of PRV from BamHI #10 through BamHI #7 after theinsertion of the swine parvovirus B gene in place of the gpX gene.

13C. Diagram of the S-PRV-020 genome showing the location of the swineparvovirus B gene inserted into the gpX region of PRV.

Restriction Enzyme Legend: B=BamHI; Ps=PstI;

Sa=SalI; N=NdeI; S=StuI; A=AccI; R=RsaI.

FIG. 14A-14C Details of S-PRV-025 construction and map data

14A. Region of S-PRV-002 starting virus showing BamHI #5 fragment. Theparvovirus B gene XbaI fragment from pSY957 is diagrammed below showinghow it will be inserted into the XbaI site by direct ligation.

14B. Region of BamHI #5 after insertion of the parvovirus B gene.

14C. Location of the parvovirus B gene inserted into both copies of therepeat in S-PRV-025.

Legend: B=BamHI; H=HindIII; X=XbaI; S=SalI; pA=glycoprotein Xpolyadenylation signal sequences; UL=unique long region; US=unique shortregion; IR=internal repeat region; TR=terminal repeat region.

FIG. 15A-15C Details of SPRV-029 Construction and Map Data

15A. Detailed map of PRV extending from BamHI #10 through BamHI #7showing the lac Z gene that will replace the gpX gene.

15B. Detailed map of PRV extending from BamHI #8' through BamHI #8 atthe junction of the unique long region and the internal repeat region(IR). The lac Z gene as a SalI fragment will replace the DNA between theStuI sites bracketing the junction.

15C. Diagram of the S-PRV-029 genome showing the locations of the lac Zgenes in the gpX region and the junction region.

Restriction Enzyme Legend: B=BamHI; Ps=PstI;

Sa=SalI; N=NdeI; S=StuI; Ba=BalI; K=KpnI.

FIG. 16 Restriction Map of Deleted S-IBR-002 EcoRI B Fragment and EcoRIF Fragment.

An 800 bp deletion including EcoRV and BglII restriction sites wasmapped in both repeat fragments.

FIG. 17 Construction of Recombinant S-IBR-004 Virus.

S-IBR-004 is an IBR recombinant virus carrying an inserted foreign gene(NEO) under the control of the PRV gpX promoter. A new XbaI site wascreated at the small unique region and the original SacI site wasdeleted.

FIG. 18 Construction of Recombinant S-IBR-008 Virus.

S-IBR-008 is a recombinant IBR virus that has a bovine rota glycoproteingene and the plasmid vector inserted in the XbaI site on the unique longregion. A site specific deletion was created at the SacI! site due tothe loss of NEO gene in the small unique region.

FIG. 19A-19C Details of HVT Construction and Map Data

19A. BamHI restriction fragment map of HVT. Fragments are numbered inorder of decreasing size; letters refer to small fragments whosecomparative size has not been determined.

19B. BamHI #16 fragment showing location of beta-galactosidase geneinsertion in S-HVT-001.

19C. BamHI #19 fragment showing location of beta-galactosidase geneinsertion.

Legend: B=BamHI; X=XhoI; H=HindIII; P=PstI; S =SalI; N=NdeI; R=EcoRI.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides a hybrid, nonprimate herpesviruscomprising DNA which includes a sequence essential for viral replicationof the hybrid, nonprimate herpesvirus, at least a portion of which ispresent in a sequence essential for replication of a naturally-occurringnonprimate herpesvirus and at least one foreign DNA sequence. Thesequence essential for viral replication of the hybrid, nonprimateherpesvirus may be derived from a naturally-occurring nonprimateherpesvirus.

The foreign DNA sequence may be adapted for expression in a host andencode an amino acid sequence. In one embodiment of the invention, theforeign DNA sequence is adapted for expression by a herpesviruspromoter. The herpesvirus promoter may be an endogenous upstreamherpesvirus promoter or an inserted upstream herpesvirus promoter.Examples of such herpesvirus promoters include, but are not limited to,the herpes simplex type ICP4 protein promoter, the herpes simplex type Ithymidine kinase promoter, the pseudorabies thymidine kinase promoter,the pseudorabies immediate early gene promoter, the pseudorabiesglycoprotein X promoter or the pseudorabies glycoprotein 92 promoter.

The amino acid sequence encoded by the foreign DNA sequence may be apolypeptide. Furthermore, the polypeptide may be a protein. In oneembodiment of the invention, the protein, when expressed in the host, isantigenic. In a further embodiment of the invention, the protein isswine rotavirus glycoprotein 38. In yet another embodiment of theinvention, the protein is bovine rotavirus glycorprotein 38. In yet afurther embodiment of the invention, the protein is swine parvovirus Bcapsid protein.

The hybrid, nonprimate herpesvirus may comprise DNA of which at least aportion is present in a sequence essential for replication of anaturally-occurring alpha-herpesvirus. The alpha-herpesvirus may be apseudorabies virus, infectious bovine rhinotracheitis virus, equineherpesvirus I, feline herpesvirus I or canine herpesvirus I.Additionally the alpha-herpesvirus may be a class D herpesvirus. Theclass D herpesvirus may be pseudorabies virus, infectious bovinerhinotracheitis virus, equine herpesvirus I, feline herpesvirus I orcanine herpesvirus I. In one embodiment of the invention, thealpha-herpesvirus is an infectious bovine rhinotracheitis virus and theforeign DNA encodes the Escherichia coli neomycin resistance gene. Thisforeign DNA sequence may also be under the control of an insertedpseudorabies virus glycoprotein X promoter. Such a virus has beenconstructed, designated S-IBR-004, and deposited on May 23, 1986 withthe American Type Culture Collection (ATCC), 12301 Parklawn Drive,Rockville, Maryland 20852 under Accession No. VR 2134.

In another embodiment of the invention, the alpha-herpesvirus is aninfectious bovine rhinotracheitis virus with a deletion in the uniqueshort sequence. Furthermore, the foreign DNA sequence may encode thebovine rotavirus glycoprotein 38 gene. This virus, designated S-IBR-008,has been constructed and deposited Jun. 16, 1986 with the ATCC underAccession No. VR 2141.

Additionally the hybrid, nonprimate herpesvirus may comprise DNA ofwhich at least a portion is present in a sequence essential forreplication of a naturally-occurring gamma-herpesvirus. Thegamma-herpesvirus may be Marek's disease virus or herpesvirus ofturkeys. Moreover the gamma-herpesvirus may be a class E herpesvirus.The class E herpesvirus may be Marek's disease virus or herpesvirus ofturkeys.

Also provided is an attenuated, nonprimate herpesvirus comprising DNAwhich includes a sequence essential for viral replication of theattenuated, nonprimate herpesvirus, at least a portion of which ispresent in a sequence essential for replication of a naturally-occurringnonprimate herpesvirus, from which at least a portion of a repeatsequence has been deleted. The sequence essential for viral replicationof the attenuated, nonprimate herpesvirus may be derived from anaturally-occurring nonprimate herpesvirus.

The deleted portion of the repeat sequence may include a portion of arepeat sequence other than a junction region or may include a junctionregion. Additionally, the deleted portion of the repeat sequence maycomprise a nonessential sequence of one repeat sequence or both repeatsequences. Furthermore at least a portion of the essential sequence of arepeat may be deleted. In one embodiment of the invention, one entirerepeat may be deleted. Moreover, a sequence not located within a repeatmay additionally be deleted. In one embodiment of the invention thedeleted sequence not located within a repeat is at least a portion of agene.

The attenuated nonprimate herpesvirus may comprise DNA at least aportion of which is present in a sequence essential for replication of anaturally-occurring alpha-herpesvirus. The alpha-herpesvirus may be apseudorabies virus, infectious bovine rhinotracheitis virus, equineherpesvirus I, feline herpesvirus I or canine herpesvirus I.Additionally, the alpha-herpesvirus may be a class D herpesvirus. Theclass D herpesvirus may be a pseudorabies virus, infectious bovinerhinotracheitis virus, equine herpesvirus I, feline herpesvirus I orcanine herpesvirus I. In one embodiment of the invention, thealpha-herpesvirus is an infectious bovine rhinotracheitis virus. Inanother embodiment of the invention, the attenuated, nonprimateherpesvirus comprises an infectious bovine rhinotracheitis virus fromwhich has been deleted at least a portion of both repeat sequences. Thisvirus has been constructed, designated S-IBR-002, and deposited underATCC Accession No. VR 2140.

Further provided is an attenuated, hybrid, nonprimate herpesviruscomprising DNA which includes a sequence essential for viral replicationof the attenuated, hybrid, nonprimate herpesvirus, at least a portion ofwhich is present in a sequence essential for replication of anaturally-occurring nonprimate, herpesvirus and at least one foreign DNAsequence. The sequence essential for viral replication of theattenuated, hybrid, nonprimate virus may be derived from anaturally-occurring nonprimate herpesvirus. Furthermore, at least aportion of a repeat sequence of the attenuated, hybrid, nonprimateherpesvirus may be deleted.

The foreign DNA sequence may be adapted for expression in a host andencode an amino acid sequence. Additionally, the foreign DNA sequencemay be adapted for expression by a herpesvirus promoter. The herpesviruspromoter may be an endogenous upstream promoter or an inserted upstreamherpesvirus promoter. The herpesvirus promoter may be the herpes simplextype ICP4 protein promoter, the herpes simplex type I thymidine kinasepromoter, the pseudorabies immediate early gene promoter, thepseudorabies glycoprotein X promoter or the pseudorabies glycoprotein 92promoter.

The amino acid sequence encoded by the foreign DNA sequence may be apolypeptide. Additionally the polypeptide may be a protein. Furthermorethe protein, when expressed in a host, may be antigenic. In oneembodiment of the invention the protein is swine rotavirus glycoprotein38. In another embodiment, the protein is bovine rotavirus glycoprotein38. In a further embodiment of the invention, the protein is swineparvovirus B capsid protein.

The attenuated, hybrid, nonprimate herpesvirus may comprise DNA, atleast a portion of which is present in a sequence essential forreplication of a naturally-occurring alpha-herpesvirus. Thealpha-herpesvirus may be a pseudorabies virus, infectious bovinerhinotracheitis virus, equine herpesvirus I, feline herpesvirus I orcanine herpesvirus I. Additionally, the alpha-herpesvirus may be a classD herpesvirus. The class D herpesvirus may be pseudorabies virus,infectious bovine rhinotracheitis virus, equine herpesvirus I, felineherpesvirus I or canine herpesvirus I.

Furthermore the attenuated, hybrid, nonprimate herpesvirus may compriseDNA, at least a portion of which is present in a sequence essential forreplication of a naturally-occurring gamma-herpesvirus. Thegamma-herpesvirus may be Marek's disease virus or herpesvirus ofturkeys. Additionally the gamma-herpesvirus may be a class Eherpesvirus. The class E herpesvirus may be Marek's disease virus orherpesvirus of turkeys.

The present invention also provides a vaccine useful for immunizing ananimal against a herpesvirus disease. This vaccine comprises aneffective immunizing amount of a hybrid, nonprimate herpesvirus of thepresent invention and a suitable carrier.

Also provided is a multivalent vaccine useful for immunizing an animalagainst at least one pathogen. This vaccine comprises an effectiveimmunizing amount of a hybrid, nonprimate herpesvirus of the presentinvention which includes a foreign DNA sequence encoding a proteinwhich, when expressed in the host, is antigenic and a suitable carrier.

Furthermore, the present invention provides a vaccine useful forimmunizing an animal against a herpesvirus disease which comprises aneffective immunizing amount of an attenuated, nonprimate herpesvirusprovided by the invention and a suitable carrier. Another vaccine usefulfor immunizing an animal against a herpesvirus disease is also provided.This vaccine comprises an effective immunizing amount of an attenuated,hybrid, nonprimate herpesvirus of the present invention and a suitablecarrier.

Moreover, a multivalent vaccine useful for immunizing an animal againstat least one pathogen is provided. This vaccine comprises an effectiveimmunizing amount of an attenuated, hybrid, nonprimate herpesvirus whichincludes at least one foreign DNA sequence encoding a protein which,when expressed in the host, is antigenic and a suitable carrier.

Methods of immunizing animals against herpesvirus diseases and methodsof immunizing an animal against at least one pathogen are provided.These methods comprise administering to the animal a suitable dose of avaccine of the present invention. The animals which may be immunizedinclude, but are not limited to, bovine animals, sheep and goats.

Methods of identifying the hybrid, nonprimate herpesviruses areprovided. In one embodiment of the invention, the foreign DNA sequencein the virus is detected. In another embodiment of the invention, thepresence of the expressed polypeptide in the host animal or host cell isdetected. In yet another embodiment of the invention, the presence ofthe expressed protein in the host animal or host cell is detected.Furthermore, methods of identifying an attenuated, hybrid, nonprimateherpesvirus of the invention are provided. In one embodiment of theinvention, the foreign DNA sequence is detected. In another embodimentof the invention, the presence of the expressed polypeptide in the hostanimal or host cell is detected. In yet a third embodiment of theinvention, the presence of the expressed protein in the host animal orhost cell is detected.

The present invention further provides a method of producing in ananimal a gene product for purposes other than immunization. This methodcomprises administering to the animal a suitable quantity of a hybrid,nonprimate herpesvirus of the present invention which includes a foreignDNA sequence adapted for expression in a host, the foreign DNA sequenceof which expresses the gene product. Additionally, a gene product may beproduced in an animal for purposes other than immunization byadministering to the animal a suitable quantity of an attenuated,hybrid, nonprimate herpesvirus which includes a foreign DNA sequenceadapted for expression in a host, the foreign DNA sequence of whichexpresses the gene product.

Methods of preparing an attenuated, hybrid, nonprimate herpesvirus ofthe present invention are also provided. One method comprises isolatingnaturally-occurring nonprimate herpesvirus viral DNA and usingrestriction enzyme digestion to produce DNA restriction fragments. Theserestriction fragments are purified by agarose gel electrophoresis toobtain specific DNA fragments which are treated with appropriateenzymes, known to those skilled in the art, to produce modified viralDNA fragments. These modified DNA fragments are capable of binding tobacterial plasmid DNA sequences. Suitable bacterial plasmids areseparately treated with appropriate restriction enzymes, known to thoseskilled in the art, to produce bacterial plasmid DNA sequences capableof binding to modified viral DNA fragments. These bacterial plasmidsequences are then combined with the modified viral DNA fragments undersuitable conditions to allow the viral DNA to bind the bacterial DNA andform a viral-bacterial plasmid.

The viral-bacterial DNA plasmid is then mapped by restriction enzymes togenerate a restriction map of the viral DNA insert. The viral-bacterialDNA plasmid is then treated with a restriction enzyme known in the artto cause at least one deletion in the viral DNA sequence of theviral-bacterial DNA plasmid. This plasmid, containing at least onedeletion in the viral DNA sequence, is transfected withnaturally-occurring nonprimate herpes viral DNA into animal cells. Theanimal cells are maintained under suitable conditions to allow thenaturally-occurring nonprimate herpesviral DNA to regenerateherpesviruses and a small percent of viruses which have recombined withthe viral-foreign DNA sequence of the viral-bacterial-foreign DNAplasmid. Some of these recombined viruses have deletions in their genomeas a result of deletions in the viral DNA insert of the plasmid. Theviruses are identified and subsequently plaque purified away from theundesired viruses.

In another embodiment of the invention, naturally-occurring nonprimateherpes viral DNA is isolated and digested with appropriate restrictionenzymes to produce viral restriction fragments. Separately, foreign DNAis digested with appropriate enzymes to produce foreign DNA restrictionfragments. The foreign DNA restriction fragments are mixed with theviral DNA restriction fragments under suitable conditions so as to allowthe fragments to join together to produce viral-foreign DNA fragments.Animal cells are transfected with the viral-foreign DNA fragments andmaintained under suitable conditions so as to allow the foreign DNAfragments to regenerate herpesviruses and a small percent of viruseswhich have included foreign DNA fragments into their genome.Herpesviruses which have included desired foreign DNA fragments intotheir genome are identified and plaque purified away from undesiredherpesviruses.

MATERIALS AND METHODS

GROWTH OF HERPESVIRUS IN TISSUE CULTURE. All of the herpesviruses underdiscussion were grown in tissue culture cells. Unless otherwise noted,the cells used were: Vero cells for PRV; MDBK cells for IBR; CEF cellsfor HVT; Crandall feline kidney cells for FHV. Vero cells are suitablefor EHV and MDCK cells are suitable for CHV.

PREPARATION OF HERPESVIRUS STOCK SAMPLES. Herpesvirus stock samples wereprepared by infecting tissue culture cells at a multiplicity ofinfection of 0.01 PFU/cell in Dulbecco's Modified Eagle Medium (DMEM)containing 2 mM glutamine, 100 units/ml penicillin, 100 units/mlstreptomycin (these components were obtained from Irvine Scientific orequivalent supplier, and hereafter are referred to as complete DMEmedium) plus 1% fetal bovine serum. After cytopathic effect wascomplete, the medium and cells were harvested and the cells werepelleted at 3000 rpm for 5 minutes in a clinical centrifuge. For allherpesviruses except HVT, the cells were resuspended in 1/10 theoriginal volume of medium, and an equal volume of 2 times autoclavedskim milk (9% skim milk powder in H₂ O wgt/vol) was added. The virussample was frozen and thawed 2 times, aliquoted, and stored frozen at-70° C. The titer was usually about 108 plaque forming units per ml. ForHVT, infected cells were resuspended in complete medium containing 20%fetal bovine serum, 10% DMSO and stored frozen at -70° C.

PREPARATION OF HERPESVIRUS DNA. For herpesvirus DNA preparation, aconfluent monolayer of tissue culture cells in a 25 cm² flask or a 60 mmpetri dish was infected with 100 microliters of virus sample in 1 mlmedium. Adsorption proceeded for 1-2 hours at 37 ° C. in a humidifiedincubator with 5% CO₂ in air. After adsorption, 4 mls of complete DMEmedium plus 1% fetal bovine serum were added. After overnightincubation, or when the cells were showing 100% cytopathic effect, thecells were scraped into the medium with a cell scraper (Costar brand).The cells and medium were centrifuged at 3000 rpm for 5 minutes in aclinical centrifuge. The medium was decanted, and the cell pellet wasgently resuspended in 0.5 ml solution containing 0.01M Tris pH 7.5, 1 mMEDTA, and 0.5% Nonidet P-40 (NP40, an ionic detergent comprising anoctyl phenol ethylene oxide condensate containing an average of 9 molesethylene oxide per molecule, purchased from Sigma Chemical Co., St.Louis, MO.). The sample was incubated at room temperature for 10minutes. Ten microliters of a stock solution of RNase A (Sigma) wereadded (stock was 10 mg/ml, boiled for 10 minutes to inactivate DNAase).The sample was centrifuged for 5 minutes at 3000 rpm in a clinicalcentrifuge to pellet nuclei. The DNA pellet was removed with a pasteurpipette or wooden stick and discarded. The supernatant fluid wasdecanted into a 1.5 ml Eppendorf tube containing 25 microliters of 20%sodium dodecyl sulfate (Sigma) and 25 microliters proteinase-K (10mg/ml; Boehringer Mannheim supplier). The sample was mixed and incubatedat 37° C. for 30-60 minutes. An equal volume of water-saturated phenolwas added and the sample was mixed on a vortex mixer for 5 minutes. Thesample was centrifuged in an Eppendorf minifuge for 5 minutes at fullspeed. The upper aqueous phase was removed to a new Eppendorf tube, andtwo volumes of -20° C. absolute ethanol were added and the tube put at-20 ° C. for 30 minutes to precipitate nucleic acid. The sample wascentrifuged in an Eppendorf centrifuge at 4° C. for 5 minutes. Thesupernatant was decanted, and the pellet was washed one time with cold80% ethanol. The pellet was dried in a lyophilizer, and rehydrated in 17microliters H₂ O. For the preparation of larger amounts of DNA, theprocedure was scaled up to start with a 850 cm² roller bottle of Verocells. The DNA was stored in H₂ O or in 0.01 M Tris pH 7.5, 1 mM EDTA at-20 ° C.

PHENOL EXTRACTION. Phenol extraction was performed on any convenientvolume of DNA sample, typically between 100 microliters to 1 ml. The DNAsample was diluted in 0.lM Tris pH 7.5, 1 mM EDTA and an equal volume ofwater saturated phenol was added. The sample was mixed briefly on avortex mixer and placed on ice for 3 minutes. After centrifugation for 3minutes in a microfuge, the aqueous layer was removed to a new tube andwas precipitated by ethanol.

ETHANOL PRECIPITATION. DNA in a sample was concentrated by ethanolprecipitation. To the DNA sample were added 1/10 volume of 3M sodiumacetate, pH 7.5 and 3 volumes of cold ethanol. The DNA was precipitatedfor 30 minutes at -70 ° C. or overnight at -20 ° C. and then pelleted bycentrifugation in the microfuge for 15 minutes at 4° C. The pellet waswashed once with 200 microliters of cold 80% ethanol and pelleted againfor 10 minutes at 4° C. After air drying or lyophilization, the pelletswere resuspended in the appropriate buffer or H₂ O.

RESTRICTION ENZYME DIGESTION. DNA was cut by restriction enzymes usingthe buffer recommended by the manufacturer (InternationalBiotechnologies Inc., New Haven, CT. (IBI), Bethesda ResearchLaboratories, Bethesda, MD. (BRL), and New England Biolabs, Beverly,MA). Whenever possible, the concentration of DNA was kept below 1microgram/50 microliters. Incubation was at 37 ° C. for 1-4 hours.

AGAROSE GEL ELECTROPHORESIS OF DNA. To visualize the restriction patternof the DNA, 5 microliters of loading buffer (5X electrophoresis buffer,0.01% bromphenol blue dye, 50 mM EDTA, and 50% glycerol) were added. Thesample was loaded into a lane in a horizontal submarine electrophoresisunit containing a 0.6% agarose gel. The electrophoresis buffer was 40 mMTris, 10 mM EDTA, adjusted to pH 7.8 with acetic acid, and with orwithout 0.5 micrograms/ml ethidium bromide. The gel was run at 40-50Vfor 18 hours, and the gel was removed and stained with 0.5 micrograms/mlethidium bromide for 30 minutes. The DNA bands were visualized on a longwavelength UV transilluminator.

PHOSPHATASE TREATMENT OF DNA. Phosphatase treatment of DNA was performedby adding 1 microliter (25 units) of calf intestinal phosphatase(Boehringer Mannheim) directly to the restriction enzyme digestionreactions and continuing the incubation for 30 minutes at 37° C. Thephosphatase was inactivated for 60 minutes at 65° C prior to phenolextraction.

POLYMERASE FILL-IN REACTION. DNA was resuspended in buffer containing 50mM Tris pH 7.4, 50 mM KC1, 5 mM MgCl₂, and 400 micromolar each of thefour deoxynucleotides. Ten units of Klenow DNA polymerase (BRL) wereadded and the reaction was allowed to proceed for 15 minutes at roomtemperature. The DNA was then phenol extracted and ethanol precipitatedas above.

EXONUCLEASE RESECTION REACTION. DNA was resuspended in 100 microlitersof 60 mM Tris pH 8.0, 0.66 mM MgCl₂, 1 mM beta-mercaptoethanol. Thesample was warmed to 30° C. for 5 minutes, and 10 units of lambdaexonuclease III (BRL) were added. At frequent time intervals (e.g. every2.5 minutes), 10 microliter aliquots were diluted into 100 microlitersof 30 mM sodium acetate pH 4.5, 250 mM NaCl, 1 mM ZnSO₄, 4micrograms/100 microliters yeast tRNA, 30 units/ 100 microliters S1nuclease. After 45 minutes at 30° C., 15 microliters of stop bufferconsisting of 625 mM Tris pH 9.0, 150 mM EDTA, 1% SDS were added. Thesamples were then phenol extracted and ethanol precipitated as above.The DNA digestion products were then analyzed and purified by agarosegel electrophoresis.

PHENOL EXTRACTION OF DNA FROM AGAROSE. DNA bands cut from low meltingpoint agarose gels were diluted to less than 0.5% agarose to a finalconcentration of 0.3 M sodium acetate. The samples were heated to 65° C.to melt the agarose and then cooled to 37° C. for 5 minutes. An equalvolume of phenol was added and the sample was phenol extracted threetimes (see PHENOL EXTRACTION). The DNA was then ethanol precipitated andthe pellet resuspended at a concentration of 3-6 fmole DNA/microliter.

LIGATION. DNA was joined together by the action of the enzyme T4 DNAligase (BRL). Ligation reactions contained 10 fmoles DNA, 20 mM Tris pH7.5, 10 mM MgCl₂, 10 mM dithiothreitol (DTT), 200 micromolar ATP, and 20units T4 DNA ligase in 10 microliters final reaction volume. Theligation was allowed to proceed for 3-16 hours at 15° C. Typically DNAfragments to be ligated together were added at an equal molar ratio.Typically two different DNA fragments were joined during ligation, butjoining of three or four different DNAs at once was also possible.

RESTRICTION MAPPING OF DNA. Restriction mapping of DNA was performed asdetailed in Maniatis et al. (1). Once it was cloned, the DNA wasdigested with a number of different restriction enzymes and the DNAswere analyzed on agarose gels and the sizes of the resulting fragmentswere measured. A double digest with two different restriction enzymeswas performed on the same DNA sample to aid in the interpretation of themaps. Another approach used was to cut the DNA with a restriction enzymethat has a single unique site in the DNA, label the end of the DNA with³² P using T4 DNA kinase or Klenow DNA polymerase (see POLYMERASEFILL-IN REACTION) and then cut the DNA with other restriction enzymes atlow temperature or for short times so that only partial digestionoccurred. The subsequent analysis of the partial digestion fragments onagarose gels served to order the restriction sites on the map. All ofthese mapping procedures are well understood by those skilled in the artand are detailed in Maniatis et al. (1). The most complete restrictionmaps can only be composed once the DNA has been sequenced, and thesequence is then analyzed by a computer searching for all the knownrestriction enzyme sites. Some of our maps have been generated fromsequence information.

SOUTHERN BLOTTING OF DNA. The general procedure for Southern blottingwas taken from Maniatis et al. (1). DNA was blotted to nitrocellulosefilters (S&S BA85) in 20× SSC (1× SSC=0.15M NaCl, 0.015M sodium citrate,pH 7.0), and prehybridized in hybridization solution consisting of 30%formamide, 1× Denhardt's solution (0.02% polyvinylpyrrolidone (PVP),0.02% bovine serum albumin (BSA), 0.02% Ficoll), 6× SSC, 50 mM NaH₂ PO₄,pH 6.8, 200 micrograms/ml salmon sperm DNA for 4-24 hours at 55 ° C.Labeled probe DNA was added that had been labelled by nick translationusing a kit from Bethesda Research Laboratories (BRL) and one ³²P-labeled nucleotide. The probe DNA was separated from theunincorporated nucleotides by NACS column (BRL) or on a Sephadex G50column (Pharmacia). After overnight hybridization at 55° C., the filterwas washed once with 2X SSC at room temperature followed by two washeswith 0.1× SSC, 0.1% sodium dodecyl sulfate (SDS) for 30 minutes at 55°C. The filter was dried and autoradiographed.

DNA TRANSFECTION FOR GENERATING RECOMBINANT VIRUS. The method is basedupon the calcium phosphate DNA precipitation procedure of Graham and Vander Eb (34) with the following modifications. For transfection intoanimal cells, 0.1-0.2 micrograms of plasmid DNA containing the foreignDNA flanked by appropriate herepesvirus cloned sequences (thehomovector) were mixed with 0.3 micrograms of intact DNA. Both DNAs werestored either in H₂ O or 0.01 M Tris pH 7.5, 1 mM EDTA and the finalvolume should be less than 0.25 ml. To the mixture was added an equalvolume of 2× HEPES buffered saline (10 g N-2-hydroxyethyl piperazineN'-2-ethanesulfonic acid (HEPES), 16 g NaCl, 0.74 g KCl, , 0.25 g Na₂HPO₄. 2H₂ O, 2 g dextrose per liter H₂ O and buffered with NaOH to pH7.4). The mixture was then diluted to 0.5 ml by the addition of theappropriate volume of 1X HEPES buffered saline (prepared by diluting theabove solution 1:1 with H₂ O). After mixing, 35 microliters of 2.2 MCaCl₂ were added to the DNA mixture and mixed. The mixture was incubatedat room temperature for 30 minutes. Medium was removed from an 80%confluent monolayer of rabbit skin cells, Vero cells, or CEF cellsgrowing in a 25 cm² flask, and the DNA mixture was added to the flaskand distributed over the cells. After a 30 minute incubation at roomtemperature, 5 mls of complete DME medium plus 10% fetal bovine serumwere added. The cells were incubated for 5 hours at 37° C. in ahumidified incubator containing 5% CO₂ in air. The medium was changed at5 hours either with or without a glycerol shock. When used, the glycerolshock consisted of removing the medium and adding DME containing 20%glycerol for 3 minutes at room temperature, followed by a wash with 10%glycerol in DME, and a wash in 5% glycerol in DME, followed by theaddition of fresh complete DME medium plus 10% fetal bovine serum. Thecells were incubated at 37° C. as above for 3-4 days until cytopathiceffect from the virus was 50-100%. Virus was harvested as describedabove for the preparation of virus stocks. This stock was referred to asa transfection stock and it was subsequently screened for recombinantvirus either with or without a selection mechanism to enrich forrecombinant plaques as described below.

DIRECT LIGATION PROCEDURE FOR GENERATING RECOMBINANT HERPESVIRUSES.Rather than using homovectors and relying upon homologous recombinationto generate recombinant virus, the technique of direct ligation wasdeveloped to insert foreign genes into herpesviruses. In this instance,the cloned foreign gene did not require flanking herpesvirus DNAsequences but only required that it have restriction sites available tocut out the foreign gene fragment from the plasmid vector. A compatiblerestriction enzyme was used to cut the herpesvirus DNA. A requirement ofthe technique was that the restriction enzyme used to cut theherepesvirus DNA must cut at a limited number of sites, preferably lessthan 3 sites. The herpesvirus DNA was mixed with a 30-fold molar excessof plasmid DNA, and the mixture was cut with the appropriate restrictionenzyme. The DNA mixture was phenol extracted and ethanol precipitated toremove restriction enzymes, and ligated together according to theligation procedure detailed above. The ligated DNA mixture was thenphenol extracted, ethanol precipitated, and resuspended in 298microliters 0.01M Tris pH 7.5, 1 mM EDTA. Forty-two microliters of 2MCaCl₂ were added, followed by an equal volume of 1× HEPES bufferedsaline (see above), and the sample was used to transfect animal cells asdescribed above.

The virus in the transfection stock was then screened for foreign DNAinserts as described below. The advantage of the direct ligationtechnique was that it required less construction of sub-clones in theplasmid state, and that the recombinant virus was present in thetransfection stock at a much higher frequency than with homologousrecombination.

HAT SELECTION OF RECOMBINANT HERPESVIRUS EXPRESSING THYMIDINE KINASE.Deletion mutants of herpesviruses which suffered deletions in thethymidine kinase (TK) gene were constructed. These PRV strains have beendesignated S-PRV-002 and S-PRV-003 and have been deposited with the ATCCunder Accession No. VR 2107 and VR 2108 respectively. These TK minus(TK-) viruses have been used as recipients for the insertion of theforeign herpes simplex type 1 (HSV-1) TK gene. One HSV-1 TK gene that wehave used contains the HSV-1 ICP4 promoter and was from B. Roizman (16).It was sub-cloned to lie between two flanking regions of PRV DNA, forexample by insertion of the TK gene into PRV BamHI #5 fragment betweenXbaI and HpaI sites. The plasmid construct was then transfected with thePRV TK- DNA to yield recombinant virus. The transfection stock wasenriched for TK-containing virus by the HAT selection proceduredescribed in (37). The transfection stock was used to infect monolayersof 143 TK- cells in 60 mm culture dishes that had been preincubated inHAT medium for 16 hours at 37° C. (HAT medium: medium 199 containing 2mM glutamine, 100 units/ml penicillin, 100 units/ml streptomycin, 10%fetal bovine serum. 5×10⁵ M hypoxanthine, 10⁻⁵ M thymidine, 5×10⁻⁶ Maminopterin). Samples of the transfection stock virus were infected intothe 143 TK- cells using 10⁻³ to 10⁻⁷ dilutions of virus. After one ortwo days at 37° C., the dishes inoculated with the highest dilution ofvirus and still showing virus plaques were harvested for virus stocks,and the selection was repeated a second time. The virus stock harvestedfrom the second HAT selection was used in a plaque assay and individualplaques were picked and tested for foreign DNA inserts as describedbelow.

BROMODEOXYURIDINE SELECTION OF RECOMBINANT HERPESVIRUS. In order toinsert a foreign gene in place of a TK gene already present in theherpesvirus genome, the foreign gene was cloned in plasmids so that itcontained the same flanking homology regions as the TK genes. Theseflanking regions could be part of the TK gene itself, or parts of theherpesvirus that flank the TK gene. In either case, the plasmid DNAcontaining the foreign gene was transfected with intact herpesvirusgenomic DNA containing the HSV-1 TK gene. The transfection stock ofrecombinant virus was grown for two selections in 143 TK- cells in thepresence of 40 micrograms/ml bromodeoxyuridine (BUDR, Sigma) in completeDME medium plus 10% fetal bovine serum. The drug BUDR is an analogue ofthymidine that is recognized by the viral enzyme thymidine kinase (TK)and is ultimately incorporated into DNA. When incorporated into the DNA,BUDR is mutagenic and lethal and thus selects against viruses that havean active TK gene. By this selection method, viruses that had exchangedtheir TK gene for a foreign gene by homologous recombination wereenriched in the population. Screening for the recombinant viruses wasthen performed by one of the techniques detailed below.

HYBRIDIZATION SCREEN FOR RECOMBINANT HERPESVIRUS. One procedure used isdescribed in (38). The technique involved doing a plaque assay on PRVunder agarose, removing the agarose once plaques had formed, and liftingthe cell monolayer from the dish onto a nitrocellulose membrane filter.The filter was then processed through the Southern procedure for DNAhybridization as detailed above. The DNA probe used in the procedure wasmade from the foreign gene that had been inserted into the virus. Thusplaques that contain the foreign gene were identified, and they werepicked from the agarose overlay that had been saved.

BLUOGAL SCREEN FOR RECOMBINANT HERPESVIRUS. When the foreign geneencoded the enzyme beta-galactosidase, the plaques that contained thegene were visualized more easily. The chemical Bluogal® (BRL) wasincorporated at the level of 200-300 micrograms/ml into the agaroseoverlay during the plaque assay, and the plaques that expressed activebeta -galactosidase turned blue. The blue plaques were then picked andpurified by further blue plaque isolations. Other foreign genes wereinserted by homologous recombination such that they replaced thebeta-galactosidase gene; in this instance non-blue plaques were pickedfor purification of the recombinant virus.

ANTIBODY SCREEN FOR RECOMBINANT HERPESVIRUS. A third method forscreening the recombinant virus stock was to look directly for theexpression of the foreign gene with antibodies. Herpesvirus plaques werespotted and picked by inserting a toothpick through the agarose abovethe plaque and scraping the plaque area on the dish. Viruses were thenrinsed from the toothpick by inserting the toothpick into a well of a96-well microtiter dish (Falcon Plastics) containing a confluentmonolayer of tissue culture cells that had been washed 3 times in DMEmedium without serum. It was important for the virus to grow withoutserum at this stage to allow the immunological procedure to work. Aftercytopathic effect was complete, the plates were put at -70° C. to freezeand lyse the cells. The medium was thawed, and the freeze/thaw procedurewas repeated a second time. Then 50-100 microliters of medium wereremoved from each well and filtered under vacuum through anitrocellulose membrane (S&S BA85) using a DotBlot® apparatus (BRL). Thefilter blots were soaked in a blocking solution of 0.01M Tris pH 7.5,0.1M NaCl, 3% bovine serum albumin at room temperature for two hourswith shaking. The filter blots were then placed in a sealable bag (SearsSeal-A-Meal or equivalent), and 10 mls of the blocking solution thatcontained 10 microliters of antibody specific for the foreign proteinwere added. After overnight incubation at room temperature with shaking,the blot was washed 3 times with 100 mls 0.01M Tris, pH 7.5, 0.1M NaCl,0.05% Tween 20 detergent (Sigma). The blot was put in another sealablebag and 10 mls blocking solution containing 10⁶ counts per minute of ¹²⁵I-protein A (New England Nuclear) were added. After allowing the proteinA to bind to the antibody for 2 hours at room temperature with shaking,the blot was washed as above, dried, and overlayed with an x-ray filmand an intensifying screen (Dupont) and autoradiographed for 1-3 days at-70° C. The film was developed by standard procedures. Virus from thepositive wells which contained the recominant virus was furtherpurified.

WESTERN BLOTTING PROCEDURE. Samples of cell lysates, positive controlsand protein standards were run on a polyacrylamide gel according to theprocedure of Laemmli (44). After electrophoresis, the gel was soaked ina transfer buffer (0.025M Tris base, 0.192M glycine, 20% methanol) plus0.1% SDS for 20 minutes. The stacking gel portion was removed and theseparation gel was placed onto Whatman 3 mm paper. A matching-sizedpiece of nitrocellulose filter was prewet in the transfer buffer andplaced onto the polyacrylamide gel to cover the gel completely and makeintimate contact. A prewet piece of Whatman 3 mm paper was placed on topof the nitrocellulose filter to create a "sandwich", and the sandwichwas placed into an electrophoretic transfer device (Biorad). Thesandwich was completely submersed in transfer buffer. Theelectrophoretic transfer was carried out for 3 hours at 250 milliamps.After transfer, the nitrocellulose filter was removed from the assemblyand placed in a dish containing 50 mls of blocking buffer (50 mg/mlbovine serum albumin, 10 mM magnesium chloride, 100 mM potassiumchloride, 1 mM calcium chloride, 10 mM imidazole pH 7.0, 0.3% Tween-20,0.02% sodium azide). The nitrocellulose blot was incubated for 1-2 hoursin the blocking buffer at room temperature on a shaker. The blot wasthen placed in a sealable bag containing 15 mls of the blocking bufferplus the specific antiserum as a probe and incubated overnight at 37° C.on a shaker. The blot was then removed from the probe solution andrinsed with 5-6 changes of phosphate buffered saline over a period of 1hour. The phosphate buffered saline was removed and 50 mls of blockingbuffer containing 5×105 cpm of ¹²⁵ I labeled protein A (Amersham) wereadded. The blot was incubated for 1 hour with the labeled protein Asolution, the labeled protein A solution was removed and the blot wasrinsed with 5-6 changes of phosphate buffered saline solution containing0.3% Tween-20. The blot was air dried and autoradiographed overnightwith an intensifying screen.

METHOD FOR cDNA CLONING SWINE ROTAVIRUS gp38 GENE Virus Growth. The OSUstrain of porcine rotavirus (ATCC VR-892) was propagated on MA-104 cells(Rhesus monkey kidney cells from MA Bioproducts). Confluent monolayerswere infected at a multiplicity of infection of greater than 10 in DMEMcontaining 5 micrograms/ml trypsin. Cells were incubated with the virusfor 48 hours or until a cytopathic effect was obtained. Media and celldebris were collected and centrifuged at 10,000× g for 20 minutes at 4°C. The supernatant containing the rotavirus was then centrifuged at10,000× g in a preparative Beckman Ti45 rotor at 4° C. Virus pelletswere resuspended in SM medium (50 mM Tris-HCl pH 7.5, 100 mM KCL, 10 mMMgCl₂) and homogenized lightly in a Dounce-type homogenizer. Theresuspended virus was centrifuged at 10,000× g for 10 minutes thenloaded onto 25-50% CsCl gradients in SM buffer. Gradients werecentrifuged at 100,000× g for 4 hours at 20° C. The two blue-white bandsrepresenting intact virions and cores of rotavirus were collected,diluted, and the CsCl gradient procedure was repeated a second time.Virus obtained from the second gradient was dialyzed overnight againstSM buffer at 4° C.

Viral RNA Isolation. Dialyzed swine rotavirus was twice extracted withan equal volume of SDS/phenol then twice more with chloroform:isoamylalcohol (24:1). The double stranded RNA was precipitated withethanol in the presence of 0.2M sodium acetate, centrifuged andresuspended in water. The yield was typically 100 micrograms from 1,000cm² of infected cells.

Synthesis and Cloning of gp38 cDNA. 160 micrograms of double-strandedswine rotavirus RNA obtained from the above procedure was mixed with onemicrogram each of two synthetic oligo nucleotide primers in a volume of160 microliters (sequences of primers were:5'-GGGAATTCTGCAGGTCACATCATACAATTCTAATCTAAG-3' and5'-GGGAATTCTGCAGGCTTTAAAAGAGAGAATTTCCGTTTGGCTA-3') derived from thepublished sequence of bovine rotavirus (24). The RNA-primer mixture wasboiled for 3 minutes in a water bath then chilled on ice. Additions of25 microliters of 1M Tris-HCl pH 8.3, 35 microliters of 1M KC1, 10microliters of 0.25M MgCl₂, 7 microliters of 0.7M 2-mercaptoethanol, 7microliters of 20 mM dNTP's and 6 microliters of reverse transcriptase(100 units) were made sequentially. The reaction was incubated at 42 °C. for 1.5 hours then 10 microliters of 0.5M EDTA pH 8.0 was added andthe solution was extracted once with chloroform: phenol (1:1). Theaqueous layer was removed and to it 250 microliters of 4M ammoniumacetate and 1.0 ml of 95% ethanol was added, the mixture was frozen indry ice and centrifuged in the cold. The resulting pellet wasresuspended in 100 microliters of 10 mM Tris-HCl pH 7.5 and the ammoniumacetate precipitation procedure was repeated. The pellet was resuspendedin 100 microliters of 0.3M KOH and incubated at room temperatureovernight then at 37° C. for 2 hours. The solution was brought toneutral pH by addition of 10 microliters of 3.0M HCl and 25 microlitersof 1.0M Tris-HCl pH 7.5. The resulting single-stranded cDNA was thenprecipitated two times by the above described ammonium acetate-ethanolprocedure. The pellet obtained was resuspended in 50 microliters of 10mM Tris-HCl pH 7.5, 100 mM NaCl, 1 mM EDTA, boiled in a water bath for 2minutes then incubated at 59° C. for 16 hours. The solution waslyophilized to a volume of 15 microliters and the resultingdouble-stranded cDNA was run on a 1.0% agarose gel (Sigma agarose TypeII). The ethidium bromide stained DNA migrating at 1,000-1,100 base pairlength was excised from the gel and electroeluted in a CBS electroeluterdevice. The solution was lyophilized, and the cDNA was resuspended in 25microliters of water. To this solution was added 2 microliters of 1.0MTris-HCl pH 7.5, 2 microliters of 1M KCl, 1 microliter of 0.25M MgCl₂, 1microliter of 20 mM dNTP's and 5 units of E. coli DNA polymerase I. Thereaction was incubated at room temperature for 15 minutes, thenchloroform/phenol extracted and ammonium acetate-ethanol precipitated asdescribed above. The resulting cDNA was tailed with dCTP using terminaldeoxynucleotide transferase (BRL buffer and enzyme used). The reactionwas stopped with 2 microliters of 0.5M EDTA, chloroform/phenol extractedand precipitated with sodium acetate in the presence of 10 micrograms ofcarrier tRNA. The resuspended cDNA was mixed with 200 ng of dGMP-tailedPst I cut pBR322 (BRL catalog #5355SA) in 200 microliters of 10 mMTris-HCl pH 7.5, 100 mM NaCl, 1 mM EDTA, heated to 65° C. for 5 minutesthen 57° C. for 2 hours. The annealed cDNA-vector pBR322 was transformedonto E. coli DH-1 cells prepared for high efficiency transformation.Colonies that showed sensitivity to ampicillin and tetracyclineresistance were grown and DNA was prepared and cut with Pst I todetermine the size of the cDNA insert. Several clones having Pst Iinserts of 1,050-1,100 base pairs were analyzed and found to haveidentical restriction enzyme digest patterns. The largest clone wasdesignated pSY565 and has been deposited with the ATCC under accessionnumber 53,340. For one of these clones, the 1,100 base pair Pst I insertwas subcloned into a M13 phage sequencing vector. The entire DNAsequence of this clone was determined and is shown in FIG. 10A and 10B.The location of the gp38 open reading frame was determined from theamino acid homology to human and bovine sequences already published(24).

METHOD FOR cDNA CLONING BOVINE ROTAVIRUS gp38 GENE Virus Growth. TheCalf Nebraska strain of bovine rotavirus (USDA) was propagated on MA-104cells (Rhesus monkey kidney cells from MA Bioproducts). Confluentmonolayers were infected at a multiplicity of infection of greater than10 in DMEM containing 5 micrograms/ml trypsin. Cells were incubated withvirus for 48 hours or until a cytopathic effect was obtained. Media andcell debris were collected and centrifuged at 10,000× g for 20 minutesat 40° C. The supernatant containing the rotavirus was then centrifugedat 10,000× g in a preparative Beckman Ti45 rotor at 4° C. Virus pelletswere resuspended in SM medium (50 mM Tris-HCl pH 7.5, 100 mM KCL, 10 mMMgCl₂) and homogenized lightly in a Dounce-type homogenizer. Theresuspended virus was centrifuged at 10,000× g for 10 minutes thenloaded onto 25-50% CsCl gradients in SM buffer. Gradients werecentrifuged at 100,000× g for 4 hours at 20° C. The two blue-white bandsrepresenting intact virions and cores of rotavirus were collected,diluted, and the CsCl gradient procedure was repeated a second time.Virus obtained from the second gradient was dialyzed overnight againstSM buffer at 4° C. Viral RNA Isolation. Dialyzed bovine rotavirus wastwice extracted with an equal volume of SDS/phenol then twice more withchloroform: isoamylalcohol (24:1). The double stranded RNA wasprecipitated with ethanol in the presence of 0.2M sodium acetate,centrifuged and resuspended in water. The yield was typically 100micrograms from 1,000 cm² of infected cells.

Synthesis and Cloning of gp38 cDNA. 160 micrograms of double-strandedbovine rotavirus RNA obtained from the above procedure was mixed withone microgram each of two synthetic oligo nucleotide primers in a volumeof 160 microliters (sequences of primers were:5'-GGGAATTCTGCAGGTCACATCATACAATTCTAATCTAAG-3' and5'-GGGAATTCTGCAGGCTTTAAAAGAGAGAATTTCOGTTTGGCTA-3') derived from thepublished sequence of bovine rotavirus (24). The RNA-primer mixture wasboiled for 3 minutes in a water bath then chilled on ice. Additions of25 microliters of 1M Tris-HCl pH 8.3, 35 microliters of 1M KC1, 10microliters of 0.25M MgCl₂, 7 microliters of 0.7M 2-mercaptoethanol, 7microliters of 20 mM dNTP's, and 6 microliters of reverse transcriptase(100 units) were made sequentially. The reaction was incubated at 42° C.for 1.5 hours then 10 microliters of 0.5M EDTA pH 8.0 was added and thesolution was extracted once with chloroform: phenol (1:1). The aqueouslayer was removed and to it 250 microliters of 4M ammonium acetate and1.0 ml of 95% ethanol was added, the mixture was frozen in dry ice andcentrifuged in the cold. The resulting pellet was resuspended in 100microliters of 10 mM Tris-HCl pH 7.5 and the ammonium acetateprecipitation procedure was repeated. The pellet was resuspended in 100microliters of 0.3M KOH and incubated at room temperature overnight thenat 37° C. for 2 hours. The solution was brought to neutral pH byaddition of 10 microliters of 3.0M HCl and 25 microliters of 1.0MTris-HCl pH 7.5.

The resulting single-stranded CDNA was then precipitated two times bythe above described ammonium acetate-ethanol procedure. The pelletobtained was resuspended in 50 microliters of 10 mM Tris-HCl pH 7.5, 100mM NaCl, 1 mM EDTA, boiled in a water bath for 2 minutes then incubatedat 59° C. for 16 hours. The solution was lyophilized to a volume of 15microliters and the resulting double-stranded CDNA was run on a 1.0%agarose gel (Sigma agarose Type II). The ethidium bromide stained DNAmigrating at 1,000-1,100 base pair length was excised from the gel andelectroeluted in a CBS electroeluter device. The solution waslyophilized, and the CDNA was resuspended in 25 microliters of water. Tothis solution was added 2 microliters of 1.0M Tris-HCl pH 7.5, 2microliters of 1M KCl, 1 microliter of 0.25M MgCl₂, 1 microliter of 20mM dNTP's, and 5 units of E. coli DNA polymerase I. The reaction wasincubated at room temperature for 15 minutes, then chloroform/phenolextracted and ammonium acetate-ethanol precipitated as described above.The resulting CDNA was tailed with dCTP using terminal deoxynucleotidetransferase (BRL buffer and enzyme used). The reaction was stopped with2 microliters of 0.5M EDTA, chloroform/phenol extracted and precipitatedwith sodium acetate in the presence of 10 micrograms of carrier tRNA.The resuspended cDNA was mixed with 200 ng of dGMP-tailed Pst I cutpBR322 (BRL catalog #5355SA) in 200 microliters of 10 mM Tris-HCl pH7.5, 100 mM NaCl, 1 mM EDTA, heated to 65° C. for 5 minutes then 57° C.for 2 hours. The annealed cDNA-vector pBR322 was transformed onto E.coli DH-1 cells prepared for high efficiency transformation. Coloniesthat showed sensitivity to ampicillin and tetracycline resistance weregrown and DNA was prepared and cut with Pst I to determine the size ofthe cDNA insert. Several clones having Pst I inserts of 1,050-1,100 basepairs were analyzed and found to have identical restriction enzymedigest patterns. For one of these clones, the 1,100 base pair Pst Iinsert was subcloned into a M13 phage sequencing vector. Part of the DNAsequence of this clone was determined and was found to be identical tothe published sequence (24).

SELECTION OF G418 RESISTANT HERPESVIRUS. The antibiotic G418 (GIBCO) hasa wide range of inhibitory activity on protein synthesis. Therecombinant virus, however, expressed the aminoglycoside3'-phosphotransferase, encoded by the NEO gene, upon acquiring theforeign gene and became resistant to G418. The transfection stocks ofrecombinant viruses were grown on MDBK (for IBR virus), Vero (for PRV)or QT35 (for HVT) cells in the presence of 500 micrograms/ml G418 incomplete DME medium plus 1% fetal bovine serum. After one or two days at37° C., plaques from the dishes inoculated with the highest dilution ofvirus were picked for virus stocks. The selection was repeated a secondor third time. The virus stocks generated from the G418 selection weretested for NEO gene insertion by the SOUTHERN BLOTTING OF DNAhybridization procedure described above.

VACCINATION STUDIES IN SWINE. Weaned pigs (4-6 weeks old) and pregnantsows were obtained from swine herds known to be free of pseudorabiesdisease. Susceptibility of the test animals to pseudorabies was furtherverified by testing the pig serum for absence of neutralizing antibodiesto pseudorabies virus (PRV). The weaned pigs and 3-to-4 day old pigletswere inoculated intramuscularly with 1 ml of virus fluid containingabout 10⁴ to 10⁶ infectious units (TCID₅₀) Animals were observed eachday after vaccination for adverse reactions (clinical signs of PRVdisease) and body temperatures were recorded. Samples of tonsillarsecretions were obtained and cultured to determined if the vaccine viruswas capable of shedding and spreading to other animals. Immunity wasdetermined by measuring PRV serum antibody levels at weekly intervalsand in some cases, by challenging the vaccinated pigs with virulentvirus. In the latter case, the vaccinated animals and a group ofnon-vaccinated pigs were inoculated with virulent, Shope strain PRV,using an amount of virus that caused PRV disease in at least 80% of theunvaccinated group of pigs. This was done about 28 days aftervaccination. The challenged animals were observed daily for signs ofdisease and for increased body temperatures. A necropsy was conducted onanimals that died and selected tissues were examined and cultured forPRV.

EXAMPLES Example 1 S-PRV-004

We have created a virus that has a deletion in the junction regionbetween the unique long DNA and the internal repeat of PRV, and adeletion in the endogenous PRV thymidine kinase gene in the unique longregion. Into the junction deletion we have cloned the herpes simplextype 1 (HSV-l) thymidine kinase (TK) gene under the control of the ICP4promoter. This virus is designated S-PRV-004.

To create this virus, we first cloned the SalI #1 fragment of PRV. PRVDNA was prepared and then cut with SalI restriction enzyme. The cut DNAwas electrophoresed on an agarose gel and the largest SailI band (15 kb)was purified from the gel (see PHENOL EXTRACTION OF DNA FROM AGAROSE).The purified DNA was ligated into the plasmid pSP64 (see LIGATION) andthe DNA mixture was used to transform E. coli HB101 according toManiatis et al. (1). The SalI #1 clone was mapped for restriction sites.

The homologous recombination procedure was used to create S-PRV-004 (seeFIG. 2). The exact position of the junction region was determined bysequencing the DNA from SalI #1 fragment. It was found that the junctionregion was positioned between two StuI sites (FIG. 2A). Two fragments ofDNA from the SalI clone were used to create the homology vector forrecombination. One was a fragment from BamHI #8' from StuI to BamHI andthe other was from BamHI #8 from BamHI to StuI (see FIGS. 1B and 2A).These fragments were cloned into the BamHI site of pSP64. This plasmidwas cut with StuI, and a 3.8 kb PvuII fragment, obtained from B. Roizman(16), The University of Chicago, and containing the ICP4 promoter on theBamHI-N fragment and the HSV-1 TK gene on the BamHI-Q fragment, fused atthe BamHI/BglII sites, was ligated into the StuI site. The net resultfrom this series of clonings was a plasmid which had suffered a deletionof 3kb from between the StuI sites, and into which 3.8kb of the foreignTK gene had been incorporated (see FIG. 2B). The TK gene was thusflanked by PRV DNA sequences to allow for insertion of the foreign geneinto the PRV genome by homologous recombination. The plasmid DNA wastranfected into rabbit skin cells along with the intact PRV DNA fromS-PRV-003, which is a pseudorabies virus that has a deletion in theendogenous TK gene. The transfection stock of virus was selected in HATmedium and the virus was identified and selected by analysis of therestriction pattern of DNA isolated from the infected cells.

S-PRV-004 contained the HSV-1 TK gene and was expressing this gene asdemonstrated by the incorporation of 14C-thymidine in a plaque assaydescribed in Tenser et al. (42) and by direct analysis of TK activity ininfected cell extracts, following the procedure of Cheng et al. (43).The location of this gene in the genome of PRV is shown in FIG. 2C.

Six weaning age pigs were vaccinated with 10⁵.0 infectious units ofS-PRV-004 and challenged with virulent PRV 28 days later, according tothe VACCINATION STUDIES IN SWINE procedure. The vaccinated pigs remainedhealthy following vaccination and developed serum neutralizing antibodyagainst PRV (see Table I below). Vaccine virus was not recovered fromnasal or tonsillar secretions. After exposure to virulent PRV, 83% ofthe vaccinated swine were protected against PRV disease.

                                      TABLE I                                     __________________________________________________________________________    RESPONSES OF WEANED PIGS VACCINATED WITH                                      S-PRV-004 AND CHALLENGED WITH VIRULENT PRV                                            Post-Vaccination   Post-Challenge                                             Antibody           Antibody                                           Antigen                                                                            Pig                                                                              Day                                                                              Day                                                                              Day                                                                              Clinical                                                                           Virus                                                                              Day                                                                              Day                                                                              Clinical                                                                           Virus                                   Level                                                                              No.                                                                              14 21 28 Signs                                                                              Isolation                                                                          7  14 Signs.sup.a                                                                        Isolation                               __________________________________________________________________________    10.sup.5.0                                                                         1  32 32 16 None None >64                                                                              >64                                                                              F    Swabs                                        2  16 32 8  None None >64                                                                              >64                                                                              F    Swabs                                        3  8  16 4  None None >64                                                                              >64                                                                              F    Swabs                                        4  4  16 8  None None >64                                                                              >64                                                                              F,C  Swabs                                        5  16 16 8  None None >64                                                                              >64                                                                              F    Swabs                                        6  8  8  4  None None >64                                                                              >64                                                                              F    Swabs                                   __________________________________________________________________________     .sup.a Key to clinical signs: C = CNS, F = Febrile                       

Example 2 S-PRV-005

S-PRV-005 is a pseudorabies virus that has a deletion in the repeatregion and in the endogenous PRV TK gene in the long unique region, andhas an insertion of the HSV-1 TK gene under the control of the ICP4promoter incorporated into both copies of the repeat region between theXbaI site and the HpaI site in the BamHI #5 fragment (See FIG. 3A-3C).

To create this virus, we first obtained a clone of BamHI #5 fragmentfrom PRV (FIG. 1B). The BamHI #5 fragment was cloned into the plasmidpACYC184 at the BamHI site (see LIGATION above). A map of the BamHI #5fragment is shown in FIG. 3A.

The plasmid containing the BaMHI #5 fragment was cut with XbaI and HpaIand the linearized plasmid was purified (see PHENOL EXTRACTION OF DNAFROM AGAROSE). The 3.8kb PvuII fragment described in Example 1 andcontaining the TK gene and ICP4 promoter was likewise purified. The XbaIsite was filled to yield a blunt end (see POLYMERASE FILL-IN REACTION),and the two DNAs were mixed and ligated together. The resulting plasmidthat had incorporated the TK gene in the XbaI-HpaI deletion was selectedand analyzed by restriction mapping (FIG. 3B).

The plasmid containing the TK gene flanked by PRV Bam HI #5 sequenceswas used to transfect rabbit skin cells along with purified DNA fromS-PRV-003, a pseudorabies virus that had a deletion in the endogenous TKgene. The resulting recombinant PRV that had incorporated the HSV-1 TKgene into the deletion in the repeats was screened and purified from thetransfection stock by the HYBRIDIZATION SCREEN FOR RECOMBINANTHERPESVIRUS procedure without any prior selection.

S-PRV-005 recombinant PRV was shown to express the HSV-1 TK gene byincorporation of ¹⁴ C-thymidine in a plaque assay (42), by analysis ofthe TK activity in infected cell lysates (43), and by immunodetection ofthe HSV-1 TK protein according to the ANTIBODY SCREEN FOR RECOMBINANTHERPESVIRUS procedure outlined above. The location of this gene in thegenome of PRV is shown in FIG. 3C.

Example 3 S-PRV-010

S-PRV-010 is a pseudorabies virus that has a deletion in the PRV TK genein the long unique region, a deletion in the repeat region, and theinsertion of the E. coli beta-galactosidase gene (lacZ gene)incorporated into both copies of the repeats at the XbaI site in BamHI#5 fragment (see FIG. 5A). The beta-galactosidase gene was constructedto be expressed using the HSV-1 TK gene promoter which we have shown inthis construct to be active in PRV.

The method used to insert the beta-galactosidase gene into S-PRV-010 wasdirect ligation (see DIRECT LIGATION PROCEDURE FOR GENERATINGRECOMBINANT HERPESVIRUS). The beta-galactosidase gene was on plasmidpJF751, obtained from Jim Hoch, Scripps Clinic and Research Foundation.This gene is truncated at the 5' end with a BamHI site that has removedthe AGT initiation codon, and the AvaI site in pBR322 was used at theother end (see FIG. 4A). The HSV-1 TK promoter (FIG. 4B) was taken fromthe McKnight TK gene as an RsaI fragment, gel purified, and ligated to asynthetic piece of DNA which contained a BamHI site within the sequenceCGGATCCG (FIG. 4C). After digestion with BamHI, the fragment was clonedinto the BamHI site at the start of the beta-galactosidase gene (FIG.4D). The plasmid was constructed with the E. coli plasmids pSP64 andpSP65 such that XbaI sites from the polylinkers could be used to excisethe entire construct from the plasmid. The ligation mixture was used totransfect E. coli HB101 according to published procedures (Maniatis etal. (1)). This construct was planned such that the first three aminoacids of the protein were from the HSV-1 TK gene, the next three werefrom the synthetic linker, and the rest were from the beta-galactosidasegene. The gene contained the following sequence at the fusion between TKand lacZ: ##STR1##

A pseudorabies virus construct designated S-PRV-002 which has a deletionin the PRV TK gene in the unique long region and a deletion in therepeat region was used as the recipient for the beta-galactosidase gene.Intact S-PRV-002 DNA was mixed with a 30-fold molar excess of plasmidDNA containing the beta-galactosidase gene under the control of theHSV-1 TK promoter, and this mixture was digested with XbaI restrictionenzyme. The ligated DNA was used to transfect animal cells, and thetransfection stock was analyzed for recombinant PRV. First, PRV DNA wasprepared from cells infected with the transfection stock virus and thisDNA was cut with restriction enzymes and analyzed on an agarose gel.This analysis showed that the recombinant virus was present as the majorspecies in the transfection stock, and it was subsequently purified fromother virus species by plaque assay coupled with the BLUOGAL SCREEN FORRECOMBINANT HERPESVIRUS. Because beta-galactosidase reacted with thedrug Bluogal® to yield a product with blue color, it was possible toplaque purify the recombinant by picking blue plaques.

The final result of the purification was the recombinant PRV designatedS-PRV-010. It was shown to express the enzyme beta-galactosidase by theformation of blue plaques as noted above, and by the detection of theenzyme in infected cell extracts using the substrateO-nitrophenyl-beta-D-galactopyranoside (Sigma) following the procedureof Norton and Coffin (35). The location of this gene in the genome ofPRV is shown in FIG. 5C.

Previous studies demonstrated that swine vaccinated with S-PRV-002developed antibody to PRV and were fully protected against clinicaldisease following exposure to virulent PRV virus. Animal studies wereconducted with S-PRV-010 to determine the utility of a recombinantpseudorabies virus as a vaccine against pseudorabies disease.

A group of weaned pigs and a litter of four-day-old piglets werevaccinated with S-PRV-010 and challenged three to four weeks later,according to VACCINATION STUDIES IN SWINE.

Responses of weaned pigs vaccinated with S-PRV-010 are shown in TableII. Administration of this virus did not cause adverse reactions in thepigs. The vaccinated animals developed PRV neutralizing antibody. Two,non-vaccinated control animals (#75 and #91) placed in contact with thevaccinates did not develop PRV antibody prior to challenge, indicatingthe vaccine virus was not shed from vaccinates. After challenge, all tenvaccinated animals remained clinically normal and free of PRV disease.In contrast, the two in-contact control animals and three of fivenon-vaccinated control animals developed PRV disease and one of thesepigs died of PRV.

To test further the utility of S-PRV-010 as a vaccine, the virus wasinoculated into 4-day old piglets. The results, presented in Table III,demonstrated that the virus elicited an antibody response in vaccinatedpiglets and did not cause adverse reactions. The virus apparently wasshed from vaccinates, since one (#67) of two non-vaccinated, in-contactcontrol piglets had developed PRV antibody by Day 24. After challenge,all vaccinated animals and the sero-positive in-contact control animalremained free of PRV disease. By comparison, the three non-vaccinatedcontrol pigs and the second in-contact control pig developed clinicalsigns of PRV and died.

The conclusion from that study is that S-PRV-010 given at a dosage of10⁴.0 or 10⁶.0 , elicits a protective response in vaccinated piglets orweaned pigs capable of preventing infection by virulent virus.

                                      TABLE II                                    __________________________________________________________________________    SEROLOGIC AND CLINICAL RESPONSES OF WEANED PIGS                               FOLLOWING VACCINATION WITH S-PRV-010 AND                                      CHALLENGE WITH WILD-TYPE PRV                                                            Antibody Titers.sup.a                                                         Post-       Post-   Post-Challenge                                  Vaccine                                                                            Pig  Vaccination Challenge                                                                             Clinical                                        GROUP                                                                              Number                                                                             Day 0                                                                             Day 14                                                                            Day 24                                                                            Day 7                                                                             Day 14                                                                            Signs                                           __________________________________________________________________________    10.sup.6.0                                                                         70   <2  64  32  32  64  None                                            Per  71   <2  16  16  16  32  None                                            Dose 72   <2  64  32  16  64  None                                                 73   <2  64  16  16  64  None                                                 74   <2  16  8   4   4   None                                                  75.sup.b                                                                          <2  <2  <2  <2  4   Depressed,                                                                    Dyspnea,                                                                      CNS Signs.sup.c                                 10.sup.4.0                                                                         76   <2  64  4   8   32  None                                            Per  77   <2  16  16  64  8   None                                            Dose 78   <2  32  16  32  8   None                                                 79   <2  8   16  64  4   None                                                 80   <2  2   <2  256 16  None                                                  81.sup.b                                                                          <2  <2  <2  <2  16  Depressed                                                                     Rhinitis,                                                                     CNS Signs                                       Con- 82   NT  NT  <2  <2  8   None                                            Trols                                                                              83   NT  NT  <2  <2  16  None                                                 84   NT  NT  <2  <2  32  CNS Signs,                                                                    Depressed,                                                                    Dyspnea                                              85   NT  NT  <2  <2  64  CNS Signs                                            86   NT  NT  <2  <2  --  CNS Signs, Died                                 __________________________________________________________________________     .sup.a Determined by RIDEA                                                    .sup.b Incontact Controls                                                     .sup.c CNS signs include Ataxia, Incoordination, Circling, Lateral            Recumbency                                                                    NT: Not Tested                                                           

                                      TABLE III                                   __________________________________________________________________________    SEROLOGIC AND CLINICAL RESPONSES OF 4-DAY-OLD PIGLETS                         FOLLOWING VACCINATION WITH S-PRV-010 AND                                      CHALLENGE WITH WILD-TYPE PRV                                                            Antibody Titers.sup.a                                                         Post-       Post-   Post-Challenge                                  Vaccine                                                                            Pig  Vaccination Challenge                                                                             Clinical                                        GROUP                                                                              Number                                                                             Day 0                                                                             Day 14                                                                            Day 24                                                                            Day 7                                                                             Day 14                                                                            Signs                                           __________________________________________________________________________    10.sup.6.0                                                                         60   <2  4   16  16  32  None                                            Per  61   <2  64  8   64  8   None                                            Dose 62   <2  32  2   16  16  None                                            10.sup.4.0                                                                         63   <2  --.sup.b                                                                          --  --  --  --                                              Per  64   <2  64  2   32  16  None                                            Dose 65   <2  2   4   32  16  None                                            In-Contact                                                                         66   <2  2   NT  --.sup.c                                                                          --  Comatose, Died                                  Controls                                                                           67   <2  <2  8   64  32  None                                            Controls                                                                           87   NT  NT  <2  --.sup.c                                                                          --  CNS Signs.sup.d,                                                              Died                                                 88   NT  NT  <2  --.sup.c                                                                          --  CNS Signs, Died                                      89   NT  NT  <2  --.sup.c                                                                          --  Died                                            __________________________________________________________________________     .sup.a Determined by RIDEA                                                    .sup.b Died 8 Days Post Vaccination From Ruptured Stomach                     .sup.c Died on or Prior to Day 7 PostChallenge                                .sup.d CNS Signs include Ataxia, Incoordination, Circling Lateral             Recumbency                                                                    NT: Not Tested                                                           

Example 4

S-PRV-007 is a pseudorabies virus that has a deletion in the PRV TK genein the unique long region, a deletion in the repeat region, and theswine rotavirus glycoprotein 38 gene under the control of the HSV-1ICP4promoter inserted into the repeat region.

S-PRV-005 virus described in Example 2 above was further engineered tocontain the rotavirus antigen (see FIGS. 6A-6C) as follows. The swinerotavirus gp38 gene was cloned into plasmid pBR322 at the PstI site byprocedures previously described herein. The resulting plasmid was calledpSY565 (see FIG. 7). The 1090 bp PstI fragment containing the gp38 genewas cloned into vector pUC4K at the PstI site such that it becameflanked by BamHI sites in a plasmid called pSY762.

Plasmid pSY590 has had a complex origin as inferred from the flow chart.These clonings were routine in nature and are of historical interest butare not strictly required to practice the invention. Briefly thishistory is:

(1) The McKnight TK gene was the HSV-1BamHI Q fragment from HaeIII at-178 relative to CAP site to BamHI at +2700 which was cloned betweenHindIII and BamHI in pBR327.

(2) pSY491..The entire TK coding region from the BglII site at +55(relative to CAP site) to the BamHI site at +2700 was cloned into theBamHI site in pSP65 and called pSY491.

(3) pSY481..The polyA signal sequence (pA) on an 800 bp SmaI fragmentfrom TK was subcloned into the SmaI site in pSP65 and was called pSY481.

(4) pSY583..The pA 800 bp SmaI fragment from pSY481 was cloned into theHincII site in pSP65 and called PSY583.

(5) pSY429..The HSV-1 BamHI N fragment was obtained from Dr. B. Roizmancloned into the BamHI site of pBR322 and was called pSY429.

(6) pSY584..The 2.2kb fragment of BamHI N from PvuII to BamHI (ref. 16)in pSY420 was subcloned into pSP65 between HincII and BamHI in thepolylinker and was called pSY584.

(7) pSY479..A plasmid was constructed from pSP64 and pSP65 thatcontained a fused polylinker sequence. Both plasmids were cut with PstIin the polylinker and PvuI in the plasmid body. The net effect of thisconstruct was to create a fusion plasmid called pSP66 which has asymmetrical polylinker sequence centered on the PstI site. pSY479 is thename of this plasmid and it also contained a PstI fragment cloned intothe PstI site that is irrelevant for the manipulations that follow.

Plasmid pSY590 was created from pSY583, pSY584, and pSY479 in a threefragment ligation of the following elements: the 3kb plasmid sequencesfrom pSY479 (pSP66) cut with PstI, the 800 bp SmaI pA fragment cut fromthe polylinker in pSY583 with PstI and BamHI, and the 2200bp BamHI Nfragment cut from pSY583 with PstI and BamHI. FIG. 7 shows the finalconfiguration of all of these DNA fragments in pSY590. There is a singleBamHI site in the plasmid between the promoter in BamHI N and the TK pAsignal that was used to insert the coding region of the gp38 gene.

For the creation of the homology vector used in the formation ofS-PRV-007, the plasmid pSY590 was opened with BamHI, and the 1090 bpgp38 gene was removed from pSY762 by cutting with BamHI, and these twofragments were ligated together to form pSY596. The correct orientationof the gp38 gene was confirmed by diagnostic restriction enzymedigestion utilizing sites with gp38 (see FIG. 10A and 10B).

In pSY596 described above, the gp38 gene resided between two flankingHSV-1 DNA fragments. These two regions were thus homologous to similarregions on the HSV-1 TK gene in S-PRV-005, and these regions were usedfor the homologous recombination to create S-PRV-007 (FIG. 6A). Theplasmid and S-PRV-005 DNAs were mixed and used in the DNA TRANSFECTIONPROCEDURE FOR GENERATING RECOMBINANT VIRUS. A virus that hadincorporated the rotavirus antigen in place of the TK gene was selectedwith BUDR. Recombinants from the selected virus stock that hadincorporated the rotavirus DNA were screened by the HYBRIDIZATION SCREENFOR RECOMBINANT HERPESVIRUS and by analyzing restriction digests of DNAby the SOUTHERN BLOTTING OF DNA procedure using the rotavirus clonedgp38 gene as probe.

The final result of this screening was a recombinant PRV calledS-PRV-007 which had the rotavirus gp38 gene incorporated into the repeatregion between the XbaI and HpaI sites in PRV BamHI #5 fragment shown inFIG. 6C. The presence in a host of gp38 expressed by S-PRV-007 has notyet been detected.

Example 5 S-PRV-012

S-PRV-012 is a pseudorabies virus that has a deletion in the PRV TKregion in the unique long region, a deletion in the repeat region, and adeletion in the unique short region encoding the PRV glycoprotein X,called gpX and identified and mapped by Rea et al. (23). The HSV-1 TKgene under the control of the ICP4 promoter was inserted in place of thegpX gene.

The following procedure was used to make the deletion of gpX and thesimultaneous insertion of the HSV-1 TK gene. The flanking regions forhomology to PRV were from cloned fragments of BamHI #10 fragment andBamHI #7 fragment extending from NdeI to BamHI (FIG. 8A-8C). The BamHIand NdeI sites were filled in according to the POLYMERASE FILL-INREACTION, and the PvuII fragment of HSV-1 DNA was inserted by LIGATION.This plasmid was transfected with intact S-PRV-002 DNA according to theDNA TRANSFECTION FOR GENERATING RECOMBINANT VIRUS procedure. Therecombinant virus was selected by HAT SELECTION OF RECOMBINANTHERPESVIRUS procedure, and screened by the ANTIBODY SCREEN FORRECOMBINANT HERPESVIRUS procedure using antibodies specific for theHSV-1 protein.

The recombinant virus selected by this procedure was designatedS-PRV-012 and has been deposited with the ATCC under Accession No.VR-2119 and was shown by RESTRICTION MAPPING OF DNA and SOUTHERNBLOTTING OF DNA to contain the HSV-1 TK gene inserted in place of thegpX gene (FIG. 8B). The ANTIBODY SCREEN FOR RECOMBINANT HERPESVIRUSprocedure showed that the virus was expressing the inserted HSV-1 TKgene. The structure of this virus is shown in FIG. 8C.

Example 6 S-PRV-013

S-PRV-013 is a pseduorabies virus that has a deletion in the TK gene inthe long unique region, a deletion in the repeat region and a deletionin the gpX coding region. The gene for E. coli beta-galactosidase (lacZgene) was inserted in place of the gpX gene and is under the control ofthe endogenous gpX gene promoter.

The following procedures were used to construct S-PRV-013 by homologousrecombination. The flanking PRV homology regions were from the clonedBamHI #10 fragment which contained the gpX promoter, and from the clonedBamHI #7 fragment extending from the NdeI site to the BamHI site (FIG.9A). The NdeI site was filled in according to the POLYMERASE FILL-INREACTION, and the beta-galactosidase gene was inserted between the BamHI#10 and BamHI #7 fragments. This construct positioned thebeta-galactosidase gene behind the gpX promoter and the gpX poly Asignal sequences with a deletion of almost all of the coding regions ofgpX. The plasmid DNA and DNA from S-PRV-002, a PRV strain with adeletion in both repeat sequences and a deletion in the thymidine kinasegene, were mixed and transfected according to the DNA TRANSFECTION FORGENERATING RECOMBINANT VIRUS procedure. The recombinant virus wasscreened and purified from the transfection stock by the BLUOGAL SCREENFOR RECOMBINANT HERPESVIRUS procedure.

The resulting virus from this screen was designated S-PRV-013 and hasbeen deposited with the ATCC under Accession No. VR 2120. It containedthe beta-galactosidase gene in place of the gpX coding regions (FIGS. 9Band 9C) as determined by PREPARATION OF HERPESVIRUS DNA followed bySOUTHERN BLOTTING OF DNA. The expression of the beta-galactosidase genewas confirmed by the BLUOGAL SCREEN FOR RECOMBINANT HERPESVIRUS test,and by the o-nitrophenyl-galactopyranoside substrate assay (27).

To confirm that the coding region for gpX had been removed fromS-PRV-013, DNA extracted from a stock of purified S-PRV-013 was digestedwith BamHI and the fragments were separated on agarose gelelectrophoresis and analyzed by SOUTHERN BLOT HYBRIDIZATION. Thehybridization probe was the BamHI-NDE fragment of pseudorabies BamHI #7fragment from the unique short region. This probe fragment included 90%of the coding sequences of gpX. In this analysis, the gpX region wasshown to be missing from S-PRV-013.

The following experiments indicate that S-PRV-013 may be used as avaccine to protect swine against psuedorabies disease. In the firststudy, susceptible weaned pigs and four-day old piglets were vaccinatedintramuscularly with S-PRV-013 as follows: 4 of each group wereinoculcated with 10⁶ TCID₅₀ and 4 were inoculated with 10⁴ TCID₅₀ ofvirus. The animals were observed, then challenged as described inVACCINATION STUDIES WITH SWINE (see Table IV below)

                                      TABLE IV                                    __________________________________________________________________________    RESPONSES OF 4-DAY-OLD PIGLETS VACCINATED                                     WITH S-PRV-013 AND CHALLENGED WITH VIRULENT PRV                                             Post-Vaccination      Post-Challenge                            Pig   Vaccine                                                                            Pig                                                                              Antibody    Clinical                                                                           Virus                                                                              Antibody                                                                              Clinical                          Group Dose No.                                                                              Day 14                                                                            Day 21                                                                            Day 28                                                                            Signs.sup.a                                                                        Isolation                                                                          Day 7                                                                             Day 14                                                                            Signs                             __________________________________________________________________________    WEANED                                                                              10.sup.6                                                                           1  4   2   2   NEG   NT.sup.b                                                                          >64 >64 NEG                                     TCID.sub.50                                                                        2  4   2   2   NEG  NT   >64 >64 NEG                                          3  2   2   2   NEG  NT   64  >64 NEG                                          4  4   2   4   NEG  NT   >64 >64 NEG                                     10.sup.4                                                                           5  2   2   2   NEG  NT   >64 >64 NEG                                     TCID.sub.50                                                                        6  <2  <2  2   NEG  NT   64  >64 NEG                                          7  <2  <2  <2  NEG  NT   64  >64 NEG                                          8  <2  <2  <2  NEG  NT   64  >64 NEG                               PIGLETS                                                                             10.sup.6                                                                           10 8   16  32  NEG  NEG  >64 >64 NEG                                     TCID.sub.50                                                                        11  --.sup.c                                                                         --  --  NEG  NEG  --  --  --                                           12 8   NT  64  NEG  NEG  >64 >64 NEG                                          13 4   8   32  NEG  NEG  >64 >64 NEG                                     10.sup.4                                                                           14 4   8   32  NEG  NEG  >64 >64 NEG                                     TCID.sub.50                                                                        15 8   16  32  NEG  NEG  >64 >64 S                                            16 2   2   8   NEG  NEG  64  >64 NEG                                          17 4   16  32  NEG  NEG  64  64  NEG                                     Contact                                                                            18 <2  <2  <2  NEG  NEG  2   16  F,C                                     Control                                                                            19  --.sup.d                                                                         --  --  NEG  NEG  --  --  --                                Challenge  20 Not Applicable        <2  --  F,C,I                             Control    21                       <2  2   F,C                                          22                       <2  2   F,C                               __________________________________________________________________________     .sup.a Key to clinical signs: NEG = Negative, C = CNS, D = Death, F =         Febrile, R = Respiratory, S = Scours                                          .sup.b Not tested                                                             .sup.c Sacrificied Day 4 postvaccination                                      .sup.d Sacrificed Day 7 postvaccination; runt doing poorly               

Following vaccination, all animals were free of adverse reactions andall but 2 (weaned pigs) developed serum neutralizing antibody titers of1:2 to 1:64. Virus was not recovered from tonsillar swabs of any pig orfrom tissues taken from the piglet (#11) sacrificed on Day 4. One of 2contact control piglets (#19) was sacrificed 7 days into the experimentbecause it was a runt and doing poorly. Tissues from this piglet werenegative when cultured for PRV. The other contact control remainedhealthy and did not develop PRV antibody prior to challenge.

After challenge, all vaccinated animals remained clinically normal anddeveloped secondary antibody responses. The contact control piglet andthe three challenge control pigs all developed typical central nervoussystem signs of PRV and one control died following challenge.

In a second study with S-PRV-013 using larger numbers of animals, 2litters of susceptible 3-day-old piglets and a group of 15 susceptibleweaned pigs were vaccinated with 10⁴ TCID₅₀ of virus, then challenged asdescribed in VACCINATION STUDIES WITH SWINE (see Tables V and VI below).

                                      TABLE V                                     __________________________________________________________________________    RESPONSES OF 3-DAY-OLD PIGLETS VACCINATED WITH                                S-PRV-013 AND CHALLENGED WITH VIRULENT PRV                                               Post-Vaccination      Post-Challenge                                          Antibody              Antibody                                             Pig                                                                              Day                                                                              Day                                                                              Day                                                                              Day                                                                              Clinical                                                                           Virus                                                                              Day                                                                              Day Clinical                                                                           Virus                            Group   No.                                                                              7  14 21 28 Signs.sup.a                                                                        Isolation                                                                          7  14  Signs                                                                              Isolation                        __________________________________________________________________________    LITTER  1  <2 2  4  4   F.sup.b                                                                           Neg  32 >64 Neg  Neg                              A       2  <2 8  8  16 F    Neg  64 >64 Neg  Neg                              VACCINATES                                                                            3  <2 8  8  16 F    Neg  16 32  Neg  Neg                                      4  <2 8  16 16 F    Neg  32 >64 Neg  Neg                                      6  <2 8  8  16 F    Neg  64 >64 Neg  Neg                              Contact 7  <2 <2 <2 <2 Neg  Neg  2  2   C,F,R                                                                              Neg                              Control 8  <2 <2 <2 <2 Neg  Neg  2  >64 C,F  Neg                              LITTER  10 <2 8  8  16 F    Neg  16 >64 Neg  Neg                              B       11 <2 8  8  16 F    Neg  32 >64 Neg  Neg                              VACCINATES                                                                            12 <2 8  32 32 F    Neg  32 >64 Neg  Neg                                      13 <2 4  16 32 F    Neg  64 >64 Neg  Neg                                      14 <2 8  16 32 Neg  Neg  64 >64 Neg  Neg                                      16 <2 4  4  16 F    Neg  32 >64 Neg  Neg                                      17 <2 8  8  32 F    Neg  64 >64 Neg  Neg                              Contact 18 <2 <2 <2 <2 Neg  Neg  2  2   C,F  Neg                              Control                                                                       CHALLENGE                                                                             19 Not      <2 Not       <2 2   C,F,R                                                                              Neg                              CONTROLS                                                                              20 Applicable                                                                             <2 Applicable                                                                              <2 2   C,F,R                                                                              Swab                                     21          <2           <2 <2  C,F,R                                                                              Swab                                     22          <2           <2 <2  C,F,R                                                                              Swab                                     23          <2           <2 Died                                                                              C,D,F,R                                                                            Swab                                                                          Tonsil,                                                                       CNS                                      24          <2           <2 <2  C,F,R                                                                              Swab                             __________________________________________________________________________     .sup.a Clinical signs: NEG = Negative, C = CNS, D = Death, F = Febrile, R     = Respiratory                                                                 .sup.b A 1° F. increase in temperature was observed in day 1 in        these vaccinates                                                         

                                      TABLE VI                                    __________________________________________________________________________    RESPONSE OF WEANED PIGS VACCINATED WITH                                       S-PRV-013 AND CHALLENGED WITH VIRULENT PRV                                               Post-Vaccination   Post-Challenge                                             Antibody           Antibody                                                Pig                                                                              Day                                                                              Day                                                                              Day                                                                              Clinical                                                                           Virus                                                                              Day                                                                              Day                                                                              Clinical                                                                           Virus                                Group   No.                                                                              0  14 21 Signs.sup.a                                                                        Isolation                                                                          7  14 Signs                                                                              Isolation                            __________________________________________________________________________    VACCINATES                                                                            35 <2 <2 4  Neg  Neg  >64                                                                              >64                                                                              Neg  Neg                                          36 <2 2  2  Neg  Neg  >64                                                                              >64                                                                              Neg  Neg                                          37 <2 2  2  Neg  Neg  >64                                                                              >64                                                                              Neg  Neg                                          38 <2 <2 2  Neg  Neg  >64                                                                              >64                                                                              Neg  Neg                                          39 <2 2  2  Neg  Neg  64 64 Neg  Neg                                          40 <2 2  4  Neg  Neg  >64                                                                              >64                                                                              Neg  Neg                                          41 <2 2  4  Neg  Neg  64 >64                                                                              Neg  Neg                                          42 <2 2  4  Neg  Neg  >64                                                                              >64                                                                              F    Neg                                          43 <2 2  2  Neg  Neg  >64                                                                              >64                                                                              F    Neg                                          44 <2 2  2  Neg  Neg  64 >64                                                                              F    Neg                                          45 <2 2  4  Neg  Neg  >64                                                                              >64                                                                              Neg  Neg                                          48 <2 2  2  Neg  Neg  >64                                                                              >64                                                                              Neg  Neg                                          47 <2 <2 2  Neg  Neg  32 >64                                                                              F    Neg                                          48 <2 2  2  Neg  Neg  >64                                                                              >64                                                                              F    Neg                                          49 <2 2  2  Neg  Neg  64 >64                                                                              F    Neg                                  CONTROLS                                                                              30 <2  NT.sup.b                                                                        <2 Not       >2 4  C,F,R                                                                              Neg                                          31 <2 NT <2 Applicable                                                                              >2 2  C,F  Neg                                          32 <2 NT <2           2  4  C,F,R                                                                              Neg                                          33 <2 NT <2           >2 Died                                                                             C,D,F,R                                                                            Tonsil                                                                        CNS                                          34 <2 NT <2           >2 4  F    Neg                                  __________________________________________________________________________     .sup.a Clinical signs: NEG = Negative, C = CNS, D = Death, F = Febrile, R     = Respiratory                                                                 .sup.b Not tested                                                        

In this experiment, all of the vaccinated animals remained healthyfollowing vaccination, developed serum neutralizing antibody to PRV anddid not shed vaccine virus in tonsillar secretions. After challenge withvirulent virus, vaccinates of both age groups remained free of PRVdisease, whereas the 3 non-vaccinated contact controls and 10 of 11 ofthe challenge controls developed severe pseudorabies disease.

Example 7 S-PRV-014

S-PRV-014 is a pseudorabies virus that has a deletion in the gpX codingregion. The gene for E. coli beta-galactosidase was inserted in place ofthe gpX gene and is under the control of the endogenous gpX promoter.The following procedures were used to create S-PRV-014 by homologousrecombination. The flanking PRV homology regions were from the clonedBamHI #10 fragment which contains the gpX promoter, and from the clonedBamHI #7 fragment extending from the NdeI site to the BamHI site (FIG.9A-9E). The NdeI site was filled in according to the POLYMERASE FILL-INREACTION, and the beta-galactosidase gene was inserted between the BamHI#10 and BamHI #7 fragments. This construct positioned thebeta-galactosidase gene behind the gpX promoter and the gpX poly Asignal sequence with a deletion of almost all of the coding region ofgpX. The plasmid DNA and DNA from wild-type PRV were mixed andtransfected according to the DNA TRANSFECTION FOR GENERATING RECOMBINANTVIRUS procedure. The recombinant virus was screened and purified fromthe transfection stock by the BLUOGAL SCREEN FOR RECOMBINANT HERPESVIRUSprocedure.

The resulting virus from this screen was designated S-PRV-014 and hasbeen deposited with the ATCC under Accession No. VR 2135. It containsthe beta-galactosidase gene in place of the gpX coding region asdetermined by PREPARATION OF HERPESVIRUS DNA followed by SOUTHERNBLOTTING DNA. The expression of the beta-galactosidase gene wasconfirmed by the BLUOGAL SCREEN FOR RECOMBINANT HERPESVIRUS test, and bythe o-nitro-phenylgalactopyranoside substrate assay (27). The structureof this virus is shown in FIG. 9D.

Example 8 S-PRV-016

S-PRV-016 is a pseudorabies virus that has a deletion in both repeatsequences, and a deletion in the gpX coding region. The gene for E. colibeta-galactosidase was inserted in place of the gpX gene and is underthe control of the endogenous gpX gene promoter.

The following procedures were used to create S-PRV-016 by homologousrecombination. The flanking PRV homology regions were from the clonedBamHI #10 fragment which contains the gpX promoter, and from the clonedBamHI #7 fragment extending from the NdeI site to the BamHI site (FIG.9A-9E). The NdeI site was filled in according to the POLYMERASE FILL-INREACTION, and the beta-galactosidase gene was inserted between the BamHI#10 and BamHI #7 fragments. The construct positioned thebeta-galactosidase gene behind the gpX promoter and the gpX poly Asignal sequence with a deletion of almost all of the coding region ofgpX. The plasmid DNA and DNA from S-PRV-001 were mixed and transfectedaccording to the DNA TRANSFECTION FOR GENERATING RECOMBINANT VIRUSprocedure. The recombinant virus was screened and purified from thetransfection stock by the BLUOGAL SCREEN FOR RECOMBINANT HERPESVIRUSprocedure.

The resulting virus from this screen was designated S-PRV-016 and hasbeen deposited with the ATCC Accession No. VR 2136. It contains thebeta-galactosidase gene in place of the gpX coding region as determinedby PREPARATION OF HERPESVIRUS DNA followed by SOUTHERN BLOTTING OF DNA.The expression of the beta-galactosidase gene was confirmed by theBLUOGAL SCREEN FOR RECOMBINANT HERPESVIRUS test, and by theo-nitro-phenylgalactopyranoside substrate assay (27). The structure ofthis virus is shown in FIG. 9E.

Example 9 S-PRV-020

S-PRV-020 is a pseudorabies virus that contains a deletion in the TKgene, a deletion in the repeat regions, and a deletion of the gpX gene,with an insertion of the swine parvovirus B capsid protein gene into thegpX region.

For cloning the swine parvovirus B gene, the NADL-8 straindouble-stranded replicative-form DNA was purified from swine parvovirusinfected cells and was supplied by Dr. T. Molitor, University ofMinnesota. The parvovirus NADL-8 DNA was cloned into the E coli plasmidpSP64 by methods detailed in (15). The DNA was partially sequenced toallow the determination of the start and the end of the major caspidprotein gene, the B gene. Identification was confirmed by comparison ofrelated sequences in the rat HI parvovirus capsid gene (29,30). Thesequence of the swine parvovirus B gene is shown in Fig. 11A and 11B.

The PRV glycoprotein X (gpX) gene promoter was used to express the Bgene and the gpX poly A signal sequence was used to terminatetranscription. The parvovirus B gene from the AccI site at nucleotide#391 to the RsaI site at nucleotide #2051 was cloned between the BamHIand NdeI site of gpX (see FIG. 7A and B). Plasmid pSY864 contained thisfragment of the parvovirus B gene flanked by the gpX signal sequence asshown in FIGS. 13A-13C It was used as the homologous DNA to promotehomologous recombination between S-PRV-013 DNA and the plasmid DNA tofacilitate the incorporation of the B gene into the PRV genome. Theplasmid DNA and S-PRV-013 DNA were mixed and transfected togetheraccording to the DNA TRANSFECTION FOR GENERATING RECOMBINANT VIRUSprocedure. The recombinant virus was screened and purified for thetransfection stock by the BLUOGAL SCREEN FOR RECOMBINANT HERPESVIRUSprocedure with the following modification. Because the parentalS-PRV-013 contained the beta-galactosidase gene, it generated blueplaques in the screening procedure. Since the parvovirus B gene wouldreplace the beta-galactosidase gene in the virus, the plaques with thisinsert would appear colorless. Therefore, colorless plaques were pickedand analyzed during this screening. A virus that contained the B genewas isolated from this screening and was designated S-PRV-020. S-PRV-020has been deposited with the ATCC under Accession No. VR 2137.

DNA from S-PRV-020 was isolated by the PREPARATION OF HERPESVIRUS DNAprocedure and used to confirm the insertion of the parvovirus B geneaccording to the SOUTHERN BLOTTING OF DNA procedure using the B gene asa probe. The test showed that the parvovirus B gene has beenincorporated into the PRV genome as expected. The structure of S-PRV-020is shown in FIG. 13C.

Example 10 S-PRV-025

The cloning of the B gene and construction of these signal sequencesonto the B gene are described in Example 9 and are shown in FIG. 12.

The DIRECT LIGATION PROCEDURE FOR GENERATING RECOMBINANT HERPESVIRUS wasused to insert the parvovirus B gene into PRV. The plasmid pSY957containing the B gene was mixed with S-PRV-002 DNA and they were cutwith restriction enzyme XbaI. The DNA mixture was ligated as describedin the method and the DNA was transfected into Vero cells. A virus thatcontained the B gene was isolated from the transfection stock of virusand was designated S-PRV-025. S-PRV-025 has been deposited with the ATCCunder Accession No. VR 2138.

DNA from S-PRV-025 was isolated by the PREPARATION OF HERPESVIRUS DNAprocedure and used to confirm the insertion of the parvovirus B geneaccording to the SOUTHERN BLOTTING OF DNA procedure using the B gene aprobe. The test showed that the parvovirus B gene has been incorporatedinto the PRV genome as expected. The structure of S-PRV-025 is shown inFIG. 14A-14C.

Example 11 S-PRV-029

S-PRV-029 is a pseudorabies virus that has a deletion in the junctionregion between the unique long region and the internal repeat of PRV,and a deletion in the gpX gene in the unique short region. The E. colibeta-galactosidase gene under the control of the gpX promoter andpolyadenylation signals has been inserted into both deletions inS-PRV-029.

To construct this virus, the SalI #1 fragment of PRV was first cloned.PRV DNA was prepared and then cut with SalI restriction enzyme. The cutDNA was electrophoresed on an agarose gel and the largest SalI band (15kb) was purified from the gel (see PHENOL EXTRACTION OF DNA FROMAGAROSE). The purified DNA was ligated into the plasmid pSP64 (seeLIGATION) and the DNA mixture was used to transform E. coli HB101according to Maniatis et al. (1). The SalI #1 clone was mapped forrestriction sites.

The homologous recombination procedure was used to create S-PRV-029. Theexact position of the junction region was determined by sequencing theDNA from the SalI #1 fragment. It was found that the junction region waspositioned between two StuI sites (see FIG. 15B). Two fragments of DNAfrom the SalI clone were used to create the homology vector forrecombination. One was a fragment from BamHI #8', from StuI to BamHI andthe other was from BamHI to StuI (FIG. 15B).

The E. coli beta-galactosidase gene was previously engineered to containthe gpX promoter and polyadenylation signals as described for S-PRV-013.To put this B-galactosidase gene into the junction region clone, aHindIII linker was first inserted into the StuI site between the BamHI#8 and BamHI #8', and into this HindIII site was cloned a HindIIIfragment containing the beta-galactosidase gene with the gpX signals.

The resulting plasmid plus wild-type PRV DNA were transfected into Verocells by the DNA TRANSFECTION FOR GENERATING RECOMBINANT VIRUSprocedure. A virus was isolated from the transfection stock thatcontained the beta-galactosidase gene inserted into both the junctiondeletion (FIG. 15B) and the gpX deletion (FIG. 15A) due to the presenceof homology to both of these regions in the plasmid. This virus waspurified by the BLUOGAL SCREEN FOR RECOMBINANT HERPESVIRUS procedure andwas designated S-PRV-029. S-PRV-029 has been deposited with the ATCCunder Accession No. VR 2139.

S-PRV-029 was shown to be expressing beta-galactosidase by the BLUOGALSCREEN FOR RECOMBINANT HERPESVIRUS procedure and theo-nitrophenylgalactopyranoside assay (27). The structure of this virusis shown in FIG. 15C.

Example 12 S-IBR-002

S-IBR-002 is an IBR virus that has a deletion of approximately 800 bp inthe repeat region of the genome. This deletion removes the only twoEcoRV restriction sites on the virus genome and an adjacent BglII site(FIG. 16).

To construct this virus, the DIRECT LIGATION PROCEDURE FOR GENERATINGRECOMBINANT HERPESVIRUSES was performed. Purified IBR DNA (Cooperstrain) digested with EcoRV restriction enzyme was mixed withDraI-restriction enzyme-digested plasmid DNA containing thebeta-galactosidase gene under the control of the HSV-1 TK promoter.After ligation the mixture was used to transfect animal cells and thetransfection stock was screened for recombinant IBR virus by theHYBRIDIZATION SCREEN FOR RECOMBINANT HERPESVIRUSES procedure. The finalresult of the purification was the recombinant IBR designated S-IBR-002.It was shown by Southern hybridization that this virus does not carryany foreign genes. Restriction enzyme analysis also showed that theinsertion sites (EcoRV) at both repeats were deleted. FIG. 16 shows therestriction map of the EcoRI B fragment which contains the EcoRVrestriction sites and the map of S-IBR-002 which lacks the EcoRV sites.S-IBR-002 has been deposited with the ATCC under Accession No. VR 2140.

Example 13 S-IBR-004

S-IBR-004 is an IBR recombinant virus carrying an inserted foreign gene,Tn5 NEO (aminoglycoside 3'-phosphotransferase) gene, under the controlof the pseudorabies virus (PRV) glycoprotein X promoter.

To construct this virus, the HindIII K DNA fragment from wild type IBRvirus was cloned into the plasmid pSP64 at the HindIII site. Thisplasmid was designated pSY524. A map of the HindIII K fragment is shownin FIG. 17. The DNA from the XhoI site to the HindIII site andcontaining the NdeI site from pSY524 was cloned into plasmid pSP65 andcalled pSY846. The NdeI to EcoRI fragment was removed from pSY846 bydigestion with NdeI and EcoRI restriction enzymes, followed byPOLYMERASE FILL-IN REACTION and LIGATION. The resulting plasmid wascalled pSY862. The plasmid pNEO (P.L. Biochemicals, Inc.) contains theaminoglycoside 3'-phosphotransferase (NEO) gene and confers resistanceto ampicillin and neomycin on E. coli hosts. The coding region of thisgene (BglII-BamHI fragment) was isolated and cloned between the PRV gpXpromoter and the HSV-Tk poly A sequence in a plasmid called pSY845.

The NEO gene construct in pSY845 was excised with HindIII, made bluntended by the POLYMERASE FILL-IN REACTION, and cloned into the SacI siteof plasmid pSY862. The final product was called pSY868.

Wild type IBR DNA was mixed with pSY868 DNA and the mixture wastransfected into rabbit skin cells to generate recombinant IBR. Therecombinant IBR virus carrying a functional NEO gene was then isolatedand purified according to the SELECTION OF G418 RESISTANT VIRUS method.

S-IBR-004 recombinant IBR was shown to express the NEO gene by the factthat cells infected with this virus were resistant to the toxicity ofG418. A detailed map of the plasmid construction is shown in FIG. 17.The structure of S-IBR-004 is also shown in FIG. 17. S-IBR-004 has beendeposited on May 23, 1986 with the ATCC under Accession No. VR 2134.

Example 14 S-IBR-008

S-IBR-008 is an IBR virus that has a deletion in the short uniqueregion, and an insertion of the bovine rotavirus glycoprotein 38 (gp38)gene in the XbaI site in the long unique region.

First the bovine rotavirus gp38 gene was engineered to containherpesvirus regulatory signals as shown in FIG. 18. This wasaccomplished by cloning the gp38 gene BamHI fragment contained inpSY1053 between the BamHI and BglII sites in pSY1052. The resultingplasmid, pSY1023, contained the PRV gpX promoter in front of the gp38gene, and the HSV-1 TK polyadenylation signal behind the gp38 gene. Theentire construct was flanked by XbaI sites to allow for the insertion ofthe XbaI fragment into IBR by direct ligation.

S-IBR-004 was the starting virus for the generation of S-IBR-008.SIBR-004 DNA and pSY1023 DNA were mixed together, cut with XbaI, andtransfected into rabbit skin cells according to the DIRECT LIGATION FORGENERATING RECOMBINANT HERPESVIRUS procedure. The transfection stock wasscreened for recombinant virus by the ANTIBODY SCREEN FOR RECOMBINANTHERPESVIRUS procedure using antibodies prepared against the rotavirusgp38 protein.

One of the viruses purified by this screen was S-IBR-008, which has thefollowing characteristics. It contains the rotavirus gp38 gene plus theplasmid DNA inserted into the XbaI site in the long unique region of thevirus genome, but no longer contains the NEO gene of parent S-IBR-004 inthe unique short region. In fact, a small deletion was created in theunique short region at the location of the NEO gene, as evidenced by theabsence of an XbaI site at this location in S-IBR-008.

S-IBR-008 was shown to be expressing the rotavirus gp38 gene by analysisof RNA transcription in infected cells, and by the ANTIBODY SCREEN FORRECOMBINANT HERPESVIRUS procedure using antibodies specific for the gp38gene. S-IBR-008 has been deposited with the ATCC under Accession No. VR2141, and its structure is shown in FIG. 18.

Example 15 Herpes Virus of Turkeys

Herpes virus of turkeys (HVT) is another herpesvirus that is similar inorganization and structure to the other animal herpesvirus examplesdescribed above. The restriction enzyme map of HVT has been published(36). This information was used as a starting point to engineer theinsertion of foreign genes into HVT. The BamHI restriction map of HVT isshown in FIG. 19A. From this data, several different regions of HVT DNAinto which insertions of foreign genes could be made were targeted. Theforeign gene chosen for insertion was the E. coli beta-galactosidasegene (beta-gal), which we have used in PRV. The promoter was the PRV gpXpromoter. The beta-gal gene was inserted into the unique long region ofHVT, specifically into the XhoI site in the BamHI #16 (3300bp) fragment,and has been shown to be expressed in an HVT recombinant by theformation of blue plaques using the substrate Bluogal. Similarly, thebeta-gal gene has been inserted into the SalI site in the repeat regioncontained within the BamHI #19 (900 bp) fragment.

These experiments show that HVT is amenable to the procedures describedwithin this application for the insertion and expression of foreigngenes in herpesviruses. In particular, two sites for insertion offoreign DNA have been identified (FIGS. 19B and 19C).

Example 16 Methods for Constructing An Attenuated Herpesvirus ContainingA Foreign DNA Insert

Applicants contemplate that the procedures disclosed herein which havebeen utilized to attenuate and insert foreign DNA sequences into PRV,IBR and HVT may be suitable for constructing other herpesviruses whichare attenuated or contain inserted foreign DNA sequences which aretranslated into amino acid sequences in a host or both. Equineherpesvirus-l (EHV) , canine herpesvirus-l (CHV), feline herpesvirus-l(FHV) or any animal herpesvirus whose genomic structure is related tothese viruses are contemplated to be amenable to these methods. Morespecifically, the following procedures may be followed to construct suchviruses.

GROW ANIMAL HERPESVIRUS IN CELL CULTURE. Established cell lines orprimary cells may be used. The methodology for the growth of theseviruses exists in the literature and does not require new art. EHV growsin Vero cells, CHV grows in Madin Darby canine kidney cells and FHVgrows in Crandell feline kidney cells.

PURIFY HERPESVIRUS DNA. The procedure disclosed herein for purifyingherpesvirus DNA was successful for all herpesviruses tested, includingPRV, IBR, HVT and cytomegalovirus, and is a general method applicable toall herpesviruses.

CLONE RESTRICTION FRAGMENTS. The cloning of herpesvirus restrictionfragments is a state of the art recombinant DNA procedure and isdescribed in Maniatis et al. (1).

MAP RESTRICTION FRAGMENTS TO GENOME. It is useful to have a restrictionenzyme map of the virus genome to identify and select regions fordeletion and insertion. Such maps are available for PRV and IBR, andpartially for HVT. A map exists for EHV, but not for CHV or FHV. Thecreation of this map does not require any new technology and is detailedin Maniatis et al. (1).

IDENTIFY RESTRICTION FRAGMENTS THAT CORRESPOND TO THE REPEAT REGION. Theidentification of repeat regions requires the SOUTHERN BLOTTINGPROCEDURE as detailed in the methods section. Clones of the repeatregion hybridize to multiple bands in a restriction enzyme digest due tothe fact that they are repeated in the virus genome. This feature,coupled with their location in the genome, are diagnostic of repeatregions.

MAKE DELETION IN REPEAT REGION CLONE. Genetic information in the repeatregion is duplicated in the other copy of the repeat in the genome.Therefore one copy of the repeat region is nonessential for replicationof the virus. Hence the repeat region is suitable for deletions andinsertions of foreign DNA. After the repeat region is cloned and mappedby restriction enzymes, enzymes may be chosen to engineer the repeatdeletion and to insert foreign DNA. It is obvious to one skilled in theart that enzyme sites will exist in a given stretch of DNA and that theycan be found by analysis. The methodology involves RESTRICTION DIGESTIONOF DNA, AGAROSE GEL ELECTROPHORESIS OF DNA, LIGATION and cloning inbacterial cells as detailed in the methods section and in Maniatis etal. (1).

MAKE INSERTION OF MARKER GENE INTO DELETION IN REPEAT REGION CLONE. Themethodology of this insertion is that described in Maniatis et al. (1)for the cloning of genes into bacteria. What is not obvious prior to thepresent disclosure is which marker genes to use that will be active in aherpesvirus, nor which signal sequences to use for the expression offoreign genes in these herpesviruses. The E. Coli beta-galactosidasegene and neomycin resistance gene under the control of the HSV-1 ICP4promoter, the PRV gpX promoter or the HSV-1 TK promoter have been used.The gpX promoter, in particular, works in PRV, IBR, and HVT. The otherpromoters have also worked in more limited testing.

TRANSFECTION WITH MARKER GENE CLONE+HERPESVIRUS DNA. The intent of thisprocedure is to put into the same cell the intact herpesvirus DNA andthe repeat region clone with the deletion and containing the markergene. Once these two DNAs are present in the same cell, normalmechanisms of homologous recombination ensure that a recombination willoccur between the homologous regions in the clone and the same region inthe herpesvirus DNA, thus substituting the marker gene for the deletedregions in the virus, with frequency of about 1%. The technique involvesthe TRANSFECTION PROCEDURE FOR GENERATING RECOMBINANT HERPESVIRUSES asdetailed in the methods section.

PURIFY HERPESVIRUS DNA. Herpesvirus DNA may be purified according to themethods described above.

SELECT RECOMBINANT PLAQUE. All the herpesviruses contemplated by thisinvention form plaques (foci of infection in cell culture) that enabletheir purification. A plaque results from infection by a single virusparticle. Thus picking a single plaque selects for the progeny of asingle recombinational event. This technical feat requires a method toidentify which plaque to pick. The methods used herein include SOUTHERNBLOTTING OF DNA to pick the plaque based upon the presence of theinserted gene, ANTIBODY SCREEN FOR RECOMBINANT HERPESVIRUS to pick theplaque based upon the presence of protein made from the gene, BLUOGALSCREEN FOR RECOMBINANT HERPESVIRUSES to pick a plaque that expresses themarker gene beta-galactosidase or G-418 SELECTION TO PURIFY RECOMBINANTHERPESVIRUSES to pick the plaque by its ability to form in the presenceof the antibiotic G-418. The first two methods are applicable to anygene; the latter two are specific for the beta-galactosidase gene andneomycin resistance gene respectively. The biology of these screeningand selection systems is such that they are applicable to anyherpesvirus, including EHV, CHV, FHV, and any animal herpesvirus relatedto them.

PURIFY RECOMBINANT VIRUS. This procedure involves multiple plaquepurifications in succession to completely purify the recombinant virusaway from the parental virus. The screening is applied at each step tochoose the plaque with which to continue. The procedures are known tothose skilled in the art of virology.

Multivalent vaccines for animals may be constructed by inserting aforeign antigen gene into a herpesvirus. The procedures and methodologyare very analogous to those used for the initial insertion of the markergene into the virus and may be performed as follows.

SUBSTITUTE FOREIGN ANTIGEN GENE FOR MARKER GENE IN REPEAT CLONE . Thisis a cloning experiment that involves putting the antigen gene behindthe same herpesvirus promoter used with the marker gene and insertingthis construction into the same identical deletion in the repeat clone.The methods for this cloning are described in Maniatis et al. (1).

TRANSFECTION WITH ANTIGEN CLONE+RECOMBINANT HERPES DNA CONTAININGMARKER. The marker gene that is already present in the herpesvirusgenome may be used to aid in the selection of the new recombinant. Forexample, it has proven useful to select white plaques instead of blueones to test for the absence of beta-galactosidase in this step. Onereason for the present of a white plaque is the replacement of thebeta-galactosidase gene with the foreign antigen gene by homologousrecombination (the desired outcome). Continued screening for this newrecombinant by the SOUTHERN BLOT PROCEDURE or by the ANTIBODY SCREEN FORRECOMBINANT HERPESVIRUS becomes more focused, less time-consuming andspecifically identifies the recombinant of interest.

ISOLATE RECOMBINANT HERPES DNA. The transfection procedure requiresintact infectious herpesvirus DNA. Recombinant herpesviruses whichinclude an inserted marker gene may be used. The isolation ofherpesvirus DNA procedure is equally applicable to these recombinantviruses.

SCREEN TRANSFECTION STOCK FOR FOREIGN GENE INSERTION. Screening methodshave been described above. They are a combination of indirect methods(screening for the absence of the marker) as well as direct methods(SOUTHERN BLOT for the antigen gene, and ANTIBODY SCREEN for theexpressed protein). The methods may be applied sequentially during thepurification of the virus.

PURIFY RECOMBINANT VIRUSES CONTAINING FOREIGN ANTIGEN GENE. Therecombinant virus containing the foreign antigen gene may be purifiedaccording to the procedures described above.

This sequence of steps, along with the methods and examples describedherein, enable anyone skilled in the art to successfully practice thisinvention with any animal herpesvirus.

Example 17

The present invention involves the use of genetically engineeredherpesviruses to protect animals against disease. It was not apparent atthe outset of research which deletions in herpesviruses would serve toattenuate the viruses to the proper degree so as to render them usefulas vaccines. Even testing vaccine candidates in animal models, e.g.mouse, does not serve as a valid indicator of the safety and efficacy ofthe vaccine in the target animal species, e.g. swine. To illustrate thispoint more clearly, Table VII shows summary data of the safety andefficacy of various pseudorabies viruses which were constructed andtested in swine according to the VACCINATION STUDIES IN SWINE procedure.

                  TABLE VII                                                       ______________________________________                                        SUMMARY OF STUDIES CONDUCTED IN                                               PIGS WITH VARIOUS PSEUDORABIES                                                VIRUS CONSTRUCTS                                                                                        Percent                                             Construct         Age     Post-Vaccination                                                                          Protection                              (Deletions/                                                                            Number   of      Antibody                                                                             Clinical                                                                             Against                               Insertions).sup.1                                                                      of Pigs  Pigs    Range  Signs  Challenge                             ______________________________________                                        S-PRV-001                                                                              9        4-6      1:32- Yes    Not Done                              (A)               weeks    >1:64 (22%)                                        S-PRV-002                                                                              12       4-6     1:4-   None   100                                   (A,B)             weeks   1:64                                                S-PRV-003                                                                              8        4-6     <1:2-  None   50                                    (B)               weeks   1:16                                                S-PRV-004                                                                              6        4-6     1:4-   None   64                                    (B,C)             weeks   1:32                                                S-PRV-010                                                                              30       4-6     <1:2-  Yes    100                                   (A,B,E)           weeks   1:16   (13%)                                                 30       3-4     1:4-   Yes    100                                                     days    1:64   (13%)                                        S-PRV-013                                                                              23       4-6     <1:2-  None   100                                   (A,B,D,E)         weeks   1:8                                                          25       3-4     1:4-   None   100                                                     days    1:64                                                S-PRV-014                                                                              5        4-6     1:4-   Yes    100                                   (D,E)             weeks   1:8    (40%)                                        S-PRV-016                                                                              5        4-6     1:4-   None   100                                   (A,D,E)           weeks   1:8                                                 ______________________________________                                         .sup.1 ARepeats; BTK; CJunction; DgpX; Ebeta-galactosidase insert        

The eight constructs that have been tested have the following deletionsand insertions in the genome of the virulent Shope strain of PRV:S-PRV-001 has a deletion in both repeat regions; S-PRV-002 has adeletion in both repeat regions and in the thymidine kinase gene;S-PRV-003 has a deletion in the thymidine kinase gene; S-PRV-004,S-PRV-010, S-PRV-013, S-PRV-014 and S-PRV-016 are described in Example#'s 1, 3, 6, 7 and 8 respectively.

A superior vaccine product must not produce clinical signs in 3-4 dayold piglets (the more sensitive age), and give 100% protection in pigsof all ages. From Table VII, it is apparent that each vaccine candidateprovided some degree of attenuation and protection in swine, but eachvaccine provided a unique response. The utility of the subjectcombinations of genomic deletions and foreign DNA insertions wasunexpected and the resulting attenuated pseudorabies viruses are bothnovel and useful as pseudorabies vaccines.

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What is claimed is:
 1. A recombinant infectious bovine rhinotracheitisvirus comprising a foreign DNA sequence encoding a polypeptide insertedinto an infectious bovine rhinotracheitis viral genome at a SacI sitewithin a 4.5Kb XhoI-HindIII subfragment of a Hind III K fraqment of theinfectious bovine rhinotracheitis viral genome, wherein expression ofthe foreign DNA sequence is under the control of a promoter locatedupstream of the foreign DNA sequence.
 2. The recombinant infectiousbovine rhinotracheitis virus of claim 1, wherein the polypeptide is anantigenic polypeptide.
 3. The recombinant infectious bovinerhinotracheitis virus of claim 1 designated S-IBR-004.
 4. Therecombinant infectious bovine rhinotracheitis virus of claim 1, whereinthe foreign DNA sequence is adapted for expression by an insertedheterologous upstream herpesvirus promoter.
 5. The recombinantinfectious bovine rhinotracheitis virus of claim 2, wherein theantigenic polypeptide is bovine rotavirus glycoprotein
 38. 6. Therecombinant infectious bovine rhinotracheitis virus of claim 4, whereinthe inserted heterologous herpesvirus promoter is selected from a groupconsisting of: the herpes simplex type 1 ICP4 protein promoter, theherpes simplex type I thymidine kinase promoter, the pseudorabiesimmediate early gene promoter, the pseudorabies glycoprotein X promoter,and the pseudorabies glycoprotein 92 promoter.
 7. A recombinantinfectious bovine rhinotracheitis virus comprising a foreign DNAsequence encoding a polypeptide inserted into an infectious bovinerhinotracheitis viral genome at a non-essential region within theHindIII K fragment of the infectious bovine rhinotracheitis viralgenome, wherein expression of the foreign DNA sequence is under thecontrol of a promoter located upstream of the foreign DNA sequence. 8.The recombinant infectious bovine rhinotracheitis virus of claim 21,wherein the non-essential region is within a 4.5 Kb XhoI to HindIIIsubfragment of the HindIII K fragment.
 9. The recombinant infectiousbovine rhinotracheitis virus of claim 1, wherein the non-essentialregion is between the SacI site and a HindIII site within the 4.5 KbXhoI to HindIII subfragment.
 10. The recombinant infectious bovinerhinotracheitis virus of claim 9, wherein the non-essential region isbetween the SacI site and a XhoI site within the 4.5 Kb XhoI to HindIIIsubfragment.